Inert format

ABSTRACT

Described herein are, proteins comprising amino acid substitutions in at least one of a first and a second polypeptide chain. Furthermore, is described the uses and methods related to said proteins.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/760,157, filed Jul. 9, 2015 (now U.S. Pat. No. 10,590,206), which isa 35 U.S.C. 371 national stage filing of International Application No.PCT/EP2014/050340, filed Jan. 9, 2014, which claims priority toInternational Application No. PCT/EP2013/064330, filed Jul. 5, 2013,U.S. Patent Application No. 61/751,045, filed Jan. 10, 2013, and DanishPatent Application No. PA201300019, filed Jan. 10, 2013. The contents ofthe aforementioned applications are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 6, 2020, is namedGMI_147USBDV_Sequence_Listing.txt and is 23,437 bytes in size.

FIELD OF INVENTION

The present invention relates to proteins, such as antibodies,comprising a first polypeptide and a second polypeptide which are inertin the sense that they do not induce any Fc receptor-mediated functionsleading to cell activation, resulting from three modifications in the Fcregion.

BACKGROUND OF THE INVENTION

The effector functions mediated by the Fc region of an antibody allowfor the destruction of foreign entities, such as the killing ofpathogens and the clearance and degradation of antigens.Antibody-dependent cell-mediated cytotoxicity (ADCC) andantibody-dependent cell-mediated phagocytosis (ADCP) are initiated bybinding of the Fc region to Fc receptor (FcR)-bearing cells, whereascomplement-dependent cytotoxicity (CDC) is initiated by binding of theFc region to C1q, which initiates the classical route of complementactivation.

Fc-mediated effector functions, such as ADCC and complement activation,have been suggested to contribute to the therapeutic efficacy ofmonoclonal antibodies used for the treatment of cancer (Weiner et al.Cell 2012, 148:1081-1084).

Previous efforts have been made to reduce unwanted effects caused bybinding to the Fc region, e.g. cytokine storm and associated toxiceffects or platelet aggregation, by providing antibody fragments orantibodies with mutated amino acid sequences. For example, antibodyfragments, such as Fab, F(ab′)₂, or scFv molecules, intrinsically lackFc-effector functions, but have a short in vivo half-life and mayrequire additional modifications to extend the half-life. Tao andMorrison (1989) describes studies of aglycosylated chimeric mouse-humanIgG. Bolt et al. (1993) describes generation of a humanized,non-mitogenic CD3 monoclonal antibody which retains in vitroimmunosuppressive properties.

Canfield and Morrison (2003) describes the binding affinity of human IgGfor its high affinity Fc receptor is determined by multiple amino acidsin the CH2 domain and is modulated by the hinge region.

Hezarah et al. (2001) describes effector function activities of a panelof mutants of a broadly neutralizing antibody against humanimmunodeficiency virus type 1.

Armour et al. (2003) describes differential binding to human FcgammaRllaand FcgammaRllb receptors by human IgG wildtype and mutant antibodies.

Idusogie et al. (2000) describes mapping of the C1q binding site onrituxan, a chimeric antibody with a human IgG1 Fc.

Shields et al (2001) describes high resolution mapping of the bindingsite on human IgG1 for FcγRI, FcγRII, RcyRIII, and FcRn and design ofIgG1 variants with improved binding to the FcγR.

Oganesyan et al. (2008) describes structural characterization of a humanFc fragment engineered for lack of effector functions.

Duncan et al. (2008) describes localization of the binding site for thehuman high-affinity Fc receptor on IgG.

Parren et al, (1992) describes the interaction of IgG subclasses withthe low affinity Fc gamma RIIa (CD32) on human monocytes, neutrophils,and platelets.

Newman et al (2001) describes modification of the Fc region of aprimatized IgG antibody to human CD4 retains its ability to modulate CD4receptors but does not deplete CD4(+) T cells in chimpanzees.

Alternatively, the hinge region of the antibody has been reported to beof importance with respect of interactions with FcγRs and complement.Dall'Acqua et al (2006) describes modulation of the effector functionsof a human IgG1 through engineering of its hinge region. However, noneof the previously engineered Fc regions are completely devoid ofFc-mediated functions. Furthermore, the impact of these specificmutations on immunogenicity and in vivo half-life is often unknown.

As described above, there is a need of proteins incapable of inducing arange of specific effector functions and at the same time have conservedpharmacokinetic properties. The present invention provides suchproteins.

SUMMARY OF INVENTION

The present invention provides proteins and antibodies having anon-activating Fc region as compared to a wild-type protein or antibody.Without being limited to theory, it is believed that the proteins andantibodies are incapable of inducing a range of effector functionscaused by interaction between the Fc region and effector components,such as Fc receptor binding.

Thus, in one aspect, the present invention relates to a proteincomprising a first polypeptide and a second polypeptide, wherein saidfirst and second polypeptide each comprises at least a hinge region, aCH2 region and a CH3 region of an immunoglobulin heavy chain, wherein inat least one of said first and second polypeptide the amino acid inpositions corresponding to positions L234, L235 and D265 in a human IgG1heavy chain, are not L, L, and D, respectively.

In another aspect, the present invention relates to a variant of aparent protein.

In another aspect, the present invention relates to a compositioncomprising a protein or variant according to the invention.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising a protein or a variant according to the inventionand a pharmaceutical acceptable carrier.

The present invention also relates to the use of a protein, variant,composition, or pharmaceutical composition according to the inventionfor use in the treatment of a disease.

Another aspect of the invention relates to a method of treatment ofcancer comprising administering a protein, variant, composition, orpharmaceutical composition according to the invention to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D: Binding curves of IgG1-CD3 (IgG1-huCLB-T3/4-F405L) orIgG1-HER2 (IgG1-HER2-169-K409R) monospecific antibody variants andbsIgG1-CD3×HER2 (IgG1-huCLB-T3/4×HER2-169) bispecific antibody variantsto their specific target on Jurkat (FIG. 1A, FIG. 1B) or AU565 cells(FIG. 1C, FIG. 1D). Data shown are mean fluorescence intensities (MFI)of one representative experiment for each cell line, as described inExample 2.

FIGS. 2A-2D: Binding curves of IgG1-CD3 (IgG1-huCLB-T3/4-F405L) orIgG1-HER2 (IgG1-HER2-169-K409R) monospecific antibody variants andbsIgG1 CD3×HER2 (IgG1-huCLB-T3/4×HER2-169) bispecific antibody variantsto their specific target on Jurkat (FIG. 2A and FIG. 2B) or AU565 cells(FIG. 2C and FIG. 2D). Data shown are mean fluorescence intensities(MFI) of one representative experiment for each cell line, as describedin Example 2.

FIGS. 3A and 3B: FACS analysis of CD69 expression on T-cells in PBMCcultures as described in Example 3. The PBMC cultures were treated withtitrated IgG1-CD3 (huCLB-T3/4) antibody variants. Representativeexamples of three experiments are shown.

FIGS. 4A and 4B: T-cell proliferation measured in ELISA as described inExample 4. PBMCs were incubated with antibody variants for three days.Representative results from two independent experiments are shown.

FIGS. 5A-5G: Induction of T-cell mediated cytotoxicity by wild-type andantibody variants (N297Q, LFLE, LFLENQ, LFLEDA, DANQ, LFLEDANQPS [FIGS.5A-5G]) was determined as described in Example 5. The averages from oneexperiment performed in duplet are shown.

FIGS. 6A-6C: Induction of T-cell mediated cytotoxicity by wild-type andantibody variants (LFLEDA, LALA [FIGS. 6A-6C]) was determined asdescribed in Example 5. The averages from two experiments performed induplet are shown.

FIGS. 7A-7I: Cytokine release in supernatant upon incubation of tumorcells and PBMCs with non-activating monospecific IgG1-CD3 or bispecificIgG-CD3×HER2 antibody variants as described in Example 5 (incubationwith GM-CSF (FIG. 7A), IFNγ (FIG. 7B), IL-1β (FIG. 7C), IL-2 (FIG. 7D),IL-6 (FIG. 7E), IL-8 (FIG. 7F), IL-10 (FIG. 7G), IL-12 (FIG. 7H), andTNFα (FIG. 7I)).

FIGS. 8A-8D: Binding of C1q to monospecific IgG1-CD3 (FIG. 8A, FIG. 8C)and bispecific IgG1-CD3×HER2 (FIG. 8B, FIG. 8D), and non-activatingantibody variants thereof was evaluated by ELISA as described in Example7. The results are representative for the experiments performed twice.

FIGS. 9A and 9B: Binding of IgG1-CD3 antibody variants (FIG. 9A) N297Q,LFLE, LFLEDA, LFLENQ, DANQ, and LFLEDANQPS and (FIG. 9B) LFLEDA and LALAto the high affinity FcγRI was evaluated by FACS analysis as describedin Example 8. Averages of two experiments are shown.

FIGS. 10A and 10B: Pharmacokinetic (PK) analysis of the non-activatingantibody variants was compared to that of wild-type IgG1-CD3 antibody asdescribed in Example 9. FIG. 10A shows the plasma concentration of humanIgG1 plotted against time. FIG. 10B shows plasma clearance ratecalculated as described in Example 9. The horizontal dotted linerepresents the average clearance rate of human IgG1 antibodies in SCIDmice (10 mL/day/kg).

FIGS. 11A and 11B: Binding curves of (FIG. 11A) monospecific antibodyvariants of IgG1-huCD3 and (FIG. 11B) bispecific antibody variantsbsIgG1 huCD3×HER2 to the human T-cell line Jurkat. Data shown are meanfluorescence intensities (MFI) of one representative experiment, asdescribed in Example 10. The tables show the antibody concentrations(μg/mL) that result in half-maximal binding (EC50).

FIGS. 12A and 12B: Binding curves of (FIG. 12A) monospecific antibodyvariants of IgG1-huCD3 and (FIG. 12B) bispecific antibody variantsbsIgG1 huCD3×HER2 to the cynomolgous T-cell line HCS-F. Data shown aremean fluorescence intensities (MFI) of one representative experiment, asdescribed in Example 10.

FIGS. 13A and 13B: IgG1-huCD3 antibody variants were titrated on PBMCs.Expression of CD69 on T-cells in PBMC culture was measured by FACSanalysis, as described in Example 11. These experiments were performedtwice and representative results from one experiment are shown.

FIGS. 14A and 14B: Human (FIG. 14A) or cynomolgous (FIG. 14B) PBMCs wereincubated with IgG1-huCD3 antibody variants for three days, after whichproliferation was measured by a cell proliferation ELISA, as describedin Example 12. Representative results from two independent experimentsare shown.

FIGS. 15A and 15B: Induction of human (FIG. 15A) and cynomolgous (FIG.15B) T-cell-mediated cytotoxicity by humanized CD3 (huCD3) antibodyvariants with non-activating LFLEDA mutations were determined asexplained in Example 13. Representative results from two independentexperiments performed in duplets are shown.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, specific modifications in amino acid positions inthe Fc region of an antibody have proven to be non-activatingmodifications making the protein inert. Specifically, it has been shownthat a particular embodiment has an in vivo plasma clearance ratecomparable to the plasma clearance rate of the wild-type antibody.

The term “non-activating” as used herein, is intended to refer to theinhibition or abolishment of the interaction of the protein according tothe invention with Fc Receptors (FcRs) present on a wide range ofeffector cells, such as monocytes, or with C1q to activate thecomplement pathway.

The term “Fc region” as used herein, is intended to refer to a regioncomprising, in the direction from the N- to C-terminal, at least a hingeregion, a CH2 region and a CH3 region.

The present invention relates in one aspect to a protein comprising afirst polypeptide and a second polypeptide, wherein said first andsecond polypeptide each comprises at least a hinge region, a CH2 regionand a CH3 region of an immunoglobulin heavy chain, wherein in at leastone of said first and second polypeptide the amino acids in thepositions corresponding to positions L234, L235 and D265 in a human IgG1heavy chain, are not L, L, and D, respectively.

The term “protein” as used herein is intended to refer to largebiological molecules comprising one or more chains of amino acids linkedto one another by peptide bonds. A single chain of amino acids may alsobe termed “polypeptide”. Thus, a protein in the context of the presentinvention may consist of one or more polypeptides. The protein accordingto the invention may be any type of protein, such as an antibody or avariant of a parent antibody.

The term “antibody” as used herein is intended to refer to animmunoglobulin molecule, a fragment of an immunoglobulin molecule, or aderivative of either thereof, which has the ability to specifically bindto an antigen under typical physiological conditions with a half-life ofsignificant periods of time, such as at least about 30 minutes, at leastabout 45 minutes, at least about one hour, at least about two hours, atleast about four hours, at least about 8 hours, at least about 12 hours,about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 ormore days, etc., or any other relevant functionally-defined period (suchas a time sufficient to induce, promote, enhance, and/or modulate aphysiological response associated with antibody binding to the antigenand/or time sufficient for the antibody to recruit an effectoractivity). The binding region (or binding domain which may be usedherein, both having the same meaning) which interacts with an antigen,comprises variable regions of both the heavy and light chains of theimmunoglobulin molecule. The constant regions of the antibodies (Abs)may mediate the binding of the immunoglobulin to host tissues orfactors, including various cells of the immune system (such as effectorcells) and components of the complement system such as C1q, the firstcomponent in the classical pathway of complement activation. Asindicated above, the term antibody herein, unless otherwise stated orclearly contradicted by context, includes fragments of an antibody thatretain the ability to specifically interact, such as bind, to theantigen. It has been shown that the antigen-binding function of anantibody may be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term “antibody”include (i) a Fab′ or Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains, or a monovalent antibody as described inWO2007059782 (Genmab A/S); (ii) F(ab′)2 fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting essentially of the VH and CH1domains; (iv) a Fv fragment consisting essentially of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,Nature 341, 544-546 (1989)), which consists essentially of a VH domainand also called domain antibodies (Holt et al; Trends Biotechnol. 2003November; 21(11):484-90); (vi) camelid or nanobodies (Revets et al;Expert Opin Biol Ther. 2005 January; 5(1):111-24) and (vii) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they may be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainantibodies or single chain Fv (scFv), see for instance Bird et al.,Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883(1988)). Such single chain antibodies are encompassed within the termantibody unless otherwise noted or clearly indicated by context.Although such fragments are generally included within the meaning ofantibody, they collectively and each independently are unique featuresof the present invention, exhibiting different biological properties andutility. These and other useful antibody fragments in the context of thepresent invention are discussed further herein. It also should beunderstood that the term antibody, unless specified otherwise, alsoincludes polyclonal antibodies, monoclonal antibodies (mAbs),antibody-like polypeptides, such as chimeric antibodies and humanizedantibodies, and antibody fragments retaining the ability to specificallybind to the antigen (antigen-binding fragments) provided by any knowntechnique, such as enzymatic cleavage, peptide synthesis, andrecombinant techniques. An antibody as generated can possess anyisotype.

When the antibody is a fragment, such as a binding fragment, it is to beunderstood within the context of the present invention that saidfragment is fused to an Fc region as herein described. Thereby, theantibody may be a fusion protein which falls within the scope of theinvention. Thus, in one embodiment, the protein is a fusion protein.

The term “immunoglobulin heavy chain” or “heavy chain of animmunoglobulin” as used herein is intended to refer to one of the heavychains of an immunoglobulin. A heavy chain is typically comprised of aheavy chain variable region (abbreviated herein as VH) and a heavy chainconstant region (abbreviated herein as CH) which defines the isotype ofthe immunoglobulin. The heavy chain constant region typically iscomprised of three domains, CH1, CH2, and CH3. The term “immunoglobulin”as used herein is intended to refer to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four potentially inter-connected by disulfide bonds. The structureof immunoglobulins has been well characterized (see for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Within the structure of the immunoglobulin, the two heavychains are inter-connected via disulfide bonds in the so-called “hingeregion”. Equally to the heavy chains each light chain is typicallycomprised of several regions; a light chain variable region (abbreviatedherein as VL) and a light chain constant region. The light chainconstant region typically is comprised of one domain, CL. Furthermore,the VH and VL regions may be further subdivided into regions ofhypervariability (or hypervariable regions which may be hypervariable insequence and/or form of structurally defined loops), also termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FRs). Each VH and VLis typically composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4 (see also Lefranc M P et al, Dev Comp ImmunolJanuary: 27(1):55-77 (2003)).

The term “full-length antibody” as used herein, refers to an antibody(e.g., a parent or variant antibody) which contains all heavy and lightchain constant and variable domains corresponding to those that arenormally found in a wild-type antibody of that isotype.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations, insertions or deletionsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, the term “human antibody”, as used herein,is not intended to include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences.

The term “first polypeptide” and “second polypeptide” as used hereinrefers to a set of polypeptides which may be identical or different inamino acid sequence. Unless otherwise stated or indicated in case of awild-type protein, the first and second polypeptides have identicalamino acid sequences.

The term “isotype” as used herein refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or anyallotypes thereof, such as IgG1m(za) and IgG1m(f)) that is encoded byheavy chain constant region genes. Thus, in one embodiment, the proteincomprises a heavy chain of an immunoglobulin of the IgG1 class or anyallotype thereof. Further, each heavy chain isotype can be combined witheither a kappa (κ) or lambda (λ) light chain, or any allotypes thereof.

The term “hinge region” as used herein refers to the hinge region of animmunoglobulin heavy chain. Thus, for example the hinge region of ahuman IgG1 antibody corresponds to amino acids 216-230 according to theEu numbering as set forth in Kabat (described in Kabat, E. A. et al.,Sequences of proteins of immunological interest. 5th Edition—USDepartment of Health and Human Services, NIH publication No. 91-3242, pp662,680,689 (1991)).

The term “CH2 region” or “CH2 domain” as used herein refers to the CH2region of an immunoglobulin heavy chain. Thus, for example the CH2region of a human IgG1 antibody corresponds to amino acids 231-340according to the Eu numbering system. However, the CH2 region may alsobe any of the other subtypes as described herein.

The term “CH3 region” or “CH3 domain” as used herein refers to the CH3region of an immunoglobulin heavy chain. Thus, for example the CH3region of a human IgG1 antibody corresponds to amino acids 341-447according to the Eu numbering system. However, the CH3 region may alsobe any of the other subtypes as described herein.

The term “amino acid corresponding to positions” as used herein refersto an amino acid position number in a human IgG1 heavy chain. Unlessotherwise stated or contradicted by context, the amino acids of theconstant region sequences are herein numbered according to the Eu-indexof numbering (described in Kabat, E. A. et al., Sequences of proteins ofimmunological interest. 5th Edition—US Department of Health and HumanServices, NIH publication No. 91-3242, pp 662,680,689 (1991)). Thus, anamino acid or segment in one sequence that “corresponds to” an aminoacid or segment in another sequence is one that aligns with the otheramino acid or segment using a standard sequence alignment program suchas ALIGN, ClustalW or similar, typically at default settings and has atleast 50%, at least 80%, at least 90%, or at least 95% identity to ahuman IgG1 heavy chain. It is considered well-known in the art how toalign a sequence or segment in a sequence and thereby determine thecorresponding position in a sequence to an amino acid position accordingto the present invention.

In the context of the present invention, the amino acid may be definedby a conservative or non-conservative class. Thus, classes of aminoacids may be reflected in one or more of the following tables:

Amino Acid Residue of Conservative Class

Acidic Residues D and E Basic Residues K, R, and H Hydrophilic UnchargedResidues S, T, N, and Q Aliphatic Uncharged Residues G, A, V, L, and INon-polar Uncharged Residues C, M, and P Aromatic Residues F, Y, and W

Alternative Physical and Functional Classifications of Amino AcidResidues

Alcohol group-containing residues S and T Aliphatic residues I, L, V,and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turnformation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q,T, K, S, G, P, D, E, and R

In the context of the present invention, a substitution in a protein isindicated as:

Original amino acid—position—substituted amino acid;

Referring to the well-recognized nomenclature for amino acids, the threeletter code, or one letter code, is used, including the codes Xaa and Xto indicate any amino acid residue. Accordingly, the notation “L234F” or“Leu234Phe” means, that the protein comprises a substitution of Leucinewith Phenylalanine in the protein amino acid position corresponding tothe amino acid in position 234 in the wild-type protein.

Substitution of an amino acid at a given position to any other aminoacid is referred to as:

Original amino acid—position; or e.g. “L234”.

For a modification where the original amino acid(s) and/or substitutedamino acid(s) may comprise more than one, but not all amino acid(s), themore than one amino acid may be separated by “,” or “/”. E.g. thesubstitution of Leucine for Phenylalanine, Arginine, Lysine orTryptophan in position 234 is:

“Leu234Phe,Arg,Lys,Trp” or “L234F,R,K,W” or “L234F/R/K/W” or “L234 to F,R, K or W”

Such designation may be used interchangeably in the context of theinvention but have the same meaning and purpose.

Furthermore, the term “a substitution” embraces a substitution into anyone of the other nineteen natural amino acids, or into other aminoacids, such as non-natural amino acids. For example, a substitution ofamino acid L in position 234 includes each of the followingsubstitutions: 234A, 234C, 234D, 234E, 234F, 234G, 234H, 234I, 234K,234M, 234N, 234Q, 234R, 234S, 234T, 234V, 234W, 234P, and 234Y. This is,by the way, equivalent to the designation 234X, wherein the X designatesany amino acid other than the original amino acid. These substitutionscan also be designated L234A, L234C, etc., or L234A,C, etc., orL234A/C/etc. The same applies by analogy to each and every positionmentioned herein, to specifically include herein any one of suchsubstitutions. It is well-known in the art when an amino acid sequencecomprises an “X” or “Xaa”, said X or Xaa represents any amino acid.Thus, X or Xaa may typically represent any of the 20 naturally occurringamino acids. The term “naturally occurring” as used herein, refers toany one of the following amino acid residues; glycine, alanine, valine,leucine, isoleucine, serine, threonine, lysine, arginine, histidine,aspartic acid, asparagine, glutamic acid, glutamine, proline,tryptophan, phenylalanine, tyrosine, methionine, and cysteine.

The terms “amino acid” and “amino acid residue” may be usedinterchangeably.

In one embodiment, in at least one of said first and second polypeptidesthe amino acid in the positions corresponding to positions L234, L235and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively,and wherein the amino acids in the positions corresponding to positionsN297 and P331 in a human IgG1 heavy chain are not Q and S, respectively.

In one embodiment, in at least one of said first and second polypeptidesthe amino acid in the positions corresponding to positions L234, L235and D265 in a human IgG1 heavy chain, is not L, L, and D, respectively,and wherein the amino acids in the positions corresponding to positionsN297 and P331 in a human IgG1 heavy chain have not been substituted. Inthis context, the term “have not been substituted” refers to the aminoacids in the amino acid positions N297 and P331 in a human IgG1 heavychain which have not been substituted with another naturally ornon-naturally occurring amino acid. Thus, a “have not been substituted”amino acid in a position corresponding to the position in a human IgG1heavy chain means the amino acid at the particular position is the sameas the naturally occurring amino acid in a human IgG1 heavy chain.

Fc-mediated effector functions form a part of the biological activity ofhuman immunoglobulin G (IgG) molecules. Examples of such effectorfunctions include e.g. antibody-dependent cell-mediated cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC) which are triggeredby the binding of various effector molecules to the Fc region. In thecontext of the present invention, “Fc binding”, “Fc Receptor binding”,“FcR binding”, and “binding of an antibody Fc region to FcR” refers tothe binding of the Fc region to an Fc Receptor (FcR) or an effectormolecule. The terms “FcγR binding” and “FcγRI binding” refers to bindingto or with an Fc region to the Fc gamma Receptor and Fc gamma ReceptorI, respectively. In some cases, when a CD3 antibody binds T-cells the Fcregion of the CD3 antibody binds to FcRs present on other cells, e.g.monocytes, which leads to activation of the T-cell. Such non-targetedactivation of T-cells may be undesired. However, targeted T-cellactivation may be highly desirable for the treatment of a range ofindications, such as cancer. Targeting of T-cells to specific cells,e.g. tumor cells, may be facilitated by use of a bispecific antibody,wherein one of the binding regions binds CD3 present on the T-cell andthe other binding region binds a target specific antigen, e.g. on atumor cell. Undesired targeted T-cell activation via Fc-mediatedcross-linking should be avoided and may be disabled by making the Fcregion inert for such activity. Thereby, interaction between said inertFc region with Fc Receptors present is prevented. An antibody of thepresent invention has been proven to be completely inert when tested inseveral different assays, i.e. see Examples 3 to 9 and 11 to 13. The CD3antibody comprising the amino acid substitutions L234F, L235E, andD265A, as described in the Examples, showed abrogation of Fc-mediatedT-cell proliferation, Fc-mediated CD69 expression on T-cells, unspecifickilling and cytokine release in a cytotoxicity assay, as well as invitro C1q binding. Similarly, the antibody comprising the amino acidsubstitutions L234F, L235E, and N297Q showed comparable results. Thus, aprotein, such as an antibody, of the present invention clearly showssuperior results in several assays when compared to a wild-type protein.

The term “inertness”, “inert” or “non-activating” as used herein, refersto an Fc region which is at least not able to bind any Fcγ Receptors,induce Fc-mediated cross-linking of FcRs, or induce FcR-mediatedcross-linking of target antigens via two Fc regions of individualproteins, such as antibodies, or is not able to bind C1q.

The term “cross-linking” as used herein, refers to the bridging of twoproteins, which may be surface proteins, by the bivalent interaction ofan antibody or the bridging of two proteins that are bound by antibodiesthrough interaction of the antibody Fc-regions with an FcR-bearing cellor the bridging of Fc Receptors to which antibodies are bound throughinteraction of the antibody with their target antigen on targetantigen-bearing cells.

The term “unspecific killing” as used herein, refers to the killing ofcells by the cytotoxic function of T-cells or other effector cells,through tumor target antigen-independent activation of said cells.

The term “proliferation” as used herein, refers to cell growth in thecontexts of cell development and cell division.

Thus, the present invention relates to a protein which does not enableFc-mediated T-cell activation, does not induce the complement system,does not bind Fcγ Receptors, but at the same time have a plasmaclearance rate which is comparable to the plasma clearance rate of awild-type protein. Such a protein may also be used in a bispecificformat.

Thus, in one embodiment, the protein has a plasma clearance rate whichdeviates from a wild-type protein by no more than 10%, such as no morethan 8%, no more than 7%, no more than 5%, no more than 3%, no more than1%, and no more than 0%.

In a particular embodiment, the protein has a plasma clearance rate(mL/day/kg) which deviates from a wild-type protein by no more than 10%,such as no more than 8%, no more than 7%, no more than 5%, no more than3%, no more than 1%, and no more than 0% wherein the plasma clearancerate is calculated by the dose (μg/kg) administered to a subject dividedby the area under the curve (AUC), wherein the AUC value is determinedfrom concentration-time curves.

In a particular embodiment, the protein has a plasma clearance ratewhich deviates from a wild-type protein by no more than 10%, such as nomore than 8%, no more than 7%, no more than 5%, no more than 3%, no morethan 1%, and no more than 0% when the plasma clearance rate (mL/day/kg)is calculated based on absorbance at 405 nm measured in a quantitativeELISA assay wherein the test samples are blood samples, such as bloodserum.

The term “quantitative ELISA” as used herein, refers to an ELISA whichallows evaluating whether a certain protein is present or absent withina sample and at the same time provides a concentration value for theprotein within the sample. In order to carry out quantitative ELISA, anaccurate standard curve must be generated to determine proteinconcentrations in the samples. A standard curve is typically a serialdilution of a known-concentration solution of the target molecule. Thus,in quantitative ELISA, the optical density (OD) of the sample iscompared to the standard curve.

In a particular embodiment, the protein has a plasma clearance ratewhich deviates from a wild-type protein by no more than 10%, such as nomore than 8%, no more than 7%, no more than 5%, no more than 3%, no morethan 1%, and no more than 0% when the plasma clearance rate (mL/day/kg)is calculated based on absorbance at 405 nm measured in a quantitativeELISA assay comprising the steps of (i) a capture antibody, such asanti-human IgG-kappa antibody, (ii) a detecting antibody recognizing theprotein, such as anti-human IgG-HRP antibody.

The term “capture antibody” as used herein refers to an unlabeledantibody which is coated to the ELISA plate. The capture antibody isused to detect/capture the protein of interest from a sample to bemeasured.

The term “detecting antibody” as used herein refers to a labeledantibody which is used to detect the protein of interest, which is boundto the capture antibody. The label consists of an enzyme that cancatalyze the conversion of a chromogenic substrate into coloredproducts. The colored product can be quantified by measuring at aspecific OD, typically around 405 nm depending on the chromogenicsubstrate.

In a particular embodiment, the protein has a plasma clearance ratewhich deviates from a wild-type protein by no more than 10%, such as nomore than 8%, no more than 7%, no more than 5%, no more than 3%, no morethan 1%, and no more than 0% when the plasma clearance rate (mL/day/kg)is calculated based on absorbance at 405 nm measured in an ELISA assaycomprising the steps of (i) coating an ELISA plate with mouse-anti-humanIgG-kappa antibody, (II) blocking with 0.2% BSA/PBS, (iii) incubatingwith dilutions of blood samples, (iv) washing the plates, (v) incubatingwith goat-anti-human IgG-HRP antibody, (vi) developing the plates with 1mg/mL 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and (vii)adding 100 μL 2% oxalic acid to stop the reaction.

The term “plasma clearance rate” as used herein, refers to aquantitative measure of the rate at which a protein is removed from theblood upon administration to a living organism. Evaluation of the plasmaclearance of the antibodies may be evaluated in SCID mice, as describedin Example 9. Thus, in one embodiment, the plasma clearance rate ismeasured by an assay comprising the steps of injecting 7-10 weeks oldC.B-17 SCID mice (CB17/Icr-Prkdcscid/IcrIcoCrl, Charles-River) with asingle i.v. dose of 100 μg (5 mg/kg) of antibody, taking blood sampleswith regular time intervals, collecting blood into heparin containingvials, centrifuging for 5 minutes at 10,000×g, coating 96-well ELISAplates overnight at 4° C. with mouse-anti-human IgG-kappa antibody inPBS, washing plates, blocking with 0.2% BSA/PBS for 1 hr at roomtemperature, incubating with dilutions (1/50 to 1/2400 in 0.2% BSA/PBST)of the blood samples or a standard curve for 1 hr at room temperature,washing plates with PBST, incubating with a goat-anti-human IgG-HRPantibody for 1 hr at room temperature, developing for about 30 min with1 mg/mL 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), adding100 μL 2% oxalic acid thereby stopping the reaction, measuringabsorbance at 405 nm in a suitable microplate reader, quantifying theantibody, plotting the antibody plasma concentration (μg/mL) over time(days) in a graph, and calculating plasma clearance rates (mL/day/kg).

The plasma clearance rate (mL/day/kg) may be calculated based on thearea under the curve (AUC) according to the following equation;

${Plasma}\mspace{14mu} {clearance}{= \frac{{Dose}\mspace{11mu} ( {{µg}\text{/}{kg}} )}{{AUC}( {{µg}\text{/}{mL}\text{/}{day}} )}}$

wherein the AUC value is determined from the concentration-time curves.

The term “deviates” as used herein when referring to the plasmaclearance rate, refers to a difference in the quantitative measure ofthe rate the protein is removed from the blood upon administration to aliving organism compared to the plasma clearance rate of a wild-typeprotein. Thus, the deviation or difference may be given as a percentagedifference.

The term “wild-type” as used herein in relation to the comparison of aprotein of the present invention, refers to a protein which is identicalto the protein of the present invention to which it is being comparedwith except for the three amino acid positions according to the presentinvention. The wild-type protein comprises the naturally occurring aminoacids of the polypeptide chains at the amino acid positions of the threemodifications of the present invention, i.e. a protein that does notcomprise the amino acid modifications according to the invention.Specifically, a wild-type antibody in relation to the inventioncomprises a Leucine at positions 234 and 235, and Aspartic acid atposition 265 when the antibody is an IgG1 antibody. Thus, a wild-typeprotein, such as an antibody, will remain an activating protein, whichis able to bind e.g. Fcγ Receptors. A wild-type protein and a proteinaccording to the invention may comprise other amino acid modificationsthan those of the invention, e.g., in order to make the proteinbispecific, or the like. Thus, “wild-type” specifically refers to theamino acids in positions corresponding to positions 234, 235 and 265 ina human IgG1 heavy chain, wherein the amino acids have not beensubstituted to any other amino acid than naturally occurring amino acidsin said positions.

The term “ELISA” as used herein refers to enzyme-linked immunosorbentassay which is a test that uses antibodies and color change to identifya substance. A first specific antibody is attached to the plate surface.Thereby the protein from a sample is added wherein binding to said firstspecific antibody is tested. A second antibody binding the protein fromthe sample is added. The second antibody is linked to an enzyme, and, inthe final step, a substance containing the enzyme's substrate is added.The subsequent reaction produces a detectable signal, most commonly acolor change in the substrate. The concept of the ELISA method iswell-known within the art and various ways of performing an ELISA arecontemplated to be part of a method to evaluate the protein according tothe invention may be evaluated with. Thus, this interpretation is not tobe understood as limiting as various forms of ELISAs may be performedsuch as described in Examples 4 or 5.

As used herein, the term “subject” is typically a human which respond tothe protein of the invention.

As can be seen in Example 9, the antibody comprising an amino acidsubstitution in the amino acid positions corresponding to L234, L235,and D265, respectively, in a human IgG1 heavy chain, showed a comparableplasma clearance rate to the wild-type antibody which was not expected.Another tested antibody comprising the amino acid substitutions L234F,L235E, and N297Q, which showed comparable results when testing T-cellactivation, T-cell proliferation, unspecific killing, cytokine releaseand C1q, did not have comparable plasma clearance rate to the wild-typeantibody. The same was observed for an antibody comprising only theamino acid substitution L234F and L235E, an antibody comprising theamino acid substitutions L234F, L235E, D265A, N297Q, and P331S, and anantibody comprising the amino acid substitutions D265A and N297Q.

In one embodiment, the first and second polypeptide is a first and asecond heavy chain of an immunoglobulin, respectively.

In the embodiments, wherein the protein is an antibody, the first andsecond polypeptides will have the same purpose and meaning as a firstand a second immunoglobulin heavy chain of an antibody. Thus, in suchembodiments, the first polypeptide and second polypeptide are to beunderstood as the first heavy chain and the second heavy chain,respectively, of the antibody.

In one embodiment, the first and second polypeptide further comprises afirst and a second binding region, respectively.

The term “binding region” as used herein, refers to a region of aprotein which is capable of binding to any molecule, such as apolypeptide, e.g. present on a cell, bacterium, or virion. The bindingregion may be a polypeptide sequence, such as a protein, protein ligand,receptor, an antigen-binding region, or a ligand-binding region capableof binding to a cell, bacterium, or virion. Specifically, the bindingregion is an antigen-binding region. If the binding region is e.g. areceptor the protein may have been prepared as a fusion protein of anFc-domain of an immunoglobulin and said receptor. If the binding regionis an antigen-binding region the protein may be an antibody, like achimeric, humanized, or human antibody or a heavy chain only antibody ora ScFv-Fc-fusion.

The term “binding” as used herein refers to the binding of a protein toa predetermined antigen or target, such as a receptor, to which bindingtypically is with an affinity corresponding to a K_(D) of about 10⁻⁶ Mor less, e.g. 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such asabout 10⁻⁹ M or less, about 10⁻¹⁰ M or less, or about 10⁻¹¹M or evenless when determined by for instance surface plasmon resonance (SPR)technology in a BIAcore 3000 instrument using the antigen as the ligandand the protein as the analyte, and binds to the predetermined antigenwith an affinity corresponding to a K_(D) that is at least ten-foldlower, such as at least 100 fold lower, for instance at least 1,000 foldlower, such as at least 10,000 fold lower, for instance at least 100,000fold lower than its affinity for binding to a non-specific antigen(e.g., BSA, casein) other than the predetermined antigen or aclosely-related antigen. The amount with which the affinity is lower isdependent on the K_(D) of the protein, so that when the K_(D) of theprotein is very low (that is, the protein is highly specific), then theamount with which the affinity for the antigen is lower than theaffinity for a non-specific antigen may be at least 10,000 fold. Theterm “K_(D)” (M), as used herein, refers to the dissociation equilibriumconstant of a particular antibody-antigen interaction.

The term “K_(D)” (M) as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction.

The term “K_(A)” (M⁻¹) as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the k_(a) by the k_(d).

The term “k_(d)” (sec⁻¹) as used herein, refers to the dissociation rateconstant of a particular antibody-antigen interaction. Said value isalso referred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹) as used herein, refers to the associationrate constant of a particular antibody-antigen interaction.

In one embodiment, the protein further comprises a first and a secondlight chain of an immunoglobulin, wherein said first light chain isconnected with said first heavy chain via disulfide bridges and saidsecond light chain is connected with said second heavy chain viadisulfide bridges, thereby forming a first binding region and a secondbinding region, respectively.

The term “disulfide bridges” as used herein refers to the covalent bondbetween two Cysteine residues, i.e. said interaction may also bedesignated a Cys-Cys interaction.

The protein according to the present invention may be monospecific,which in the context of the present invention refers to a protein whichbinds to the same epitope with its binding regions. However, theinvention is not limited to monospecific proteins but also relates tomultispecific proteins, such as bispecific proteins. Thus, in oneembodiment, at least one of the first and second binding regions bindCD3. In a particular embodiment, said first binding region binds CD3 andsaid second binding region binds any other target of interest. Suchother target may be a tumor-specific target or a cancer-specific target.

The term “human CD3” as used herein, refers to the human Cluster ofDifferentiation 3 which is part of the T-cell co-receptor proteincomplex and is composed of four distinct chains. In mammals, the complexcontains a CD3γ (gamma) chain (human CD3γ chain 182 amino acids,Swissprot P09693, and cyno CD3γ 181 amino acids, Swissprot Q95LI7), aCD3δ (delta) chain (171 amino acids, human CD3δ Swissprot P04234 SEQ IDNO:14, and cyno CD3δ Swissprot Q95LI8), two CD3ε (epsilon) chains (humanCD3ε 207 amino acids, Swissprot P07766, mature human CD3 epsilon SEQ IDNO:13; cyno CD3ε 198 amino acids, Swissprot Q95LI5, mature cyno CD3epsilon SEQ ID NO:21), and a CD3ζ-chain (zeta) chain (human CD3ζ 164amino acids, Swissprot P20963, cyno CD3ζ 166 amino acids, SwissprotQ09TKO). These chains associate with a molecule known as the T-cellreceptor (TCR) and generate an activation signal in T lymphocytes. TheTCR and CD3 molecules together comprise the TCR complex.

In a particular embodiment, at least one of said first and secondbinding region binds the epsilon chain of CD3, such as the epsilon chainof human CD3 (SEQ ID NO:14). In yet another particular embodiment, atleast one of said first and second binding region binds an epitopewithin amino acids 1-27 of the N-terminal part of mature human CD3ε(epsilon) (amino acid 1-27 of mature human CD3 epsilon as set forth inSEQ ID NO:14). In such a particular embodiment, the protein may evenfurther cross-react with other non-human primate species, such ascynomolgus monkeys (mature cyno CD3 epsilon as set forth in SEQ IDNO:21) and rhesus monkeys.

The term “mature” as used herein, refers to a protein which does notcomprise any signal or leader sequence. It is well-known to the skilledperson to determine a mature protein, or how to identify the sequence ofthe mature protein.

In one particular embodiment, the amino acids in positions correspondingto L234, L235, and D265 in a human IgG1 heavy chain of at least saidfirst polypeptide are not L, L, and D, respectively, and wherein saidfirst binding region binds CD3.

In one embodiment, both the first and second binding regions bind CD3.

The inventors of the present invention have evaluated Fcγ receptorbinding, Fc binding of complement and further additional factors whichare relevant for assessing the inertness of a protein, such as anantibody. In the case of anti-CD3 antibodies, one of such additionalfactors is the expression level of the T-cell activation marker CD69which is the earliest inducible cell surface glycoprotein requiredduring lymphoid activation.

Thus, in one embodiment, the protein of the invention, when present as amonospecific antibody binding CD3, mediates reduced Fc-mediated T-cellactivation compared to a wild-type protein by at least 50%, such as atleast 60%, at least 70%, at least 80%, at least 90%, at least 99% and100%, when the T-cell activation is determined by CD69 expression. Theterm “when present as a monospecific antibody binding CD3” refer to suchcharacteristics of the protein which are observed when the protein istested in said assay as a monospecific antibody. However, it should notbe understood as limiting the protein of the present invention to amonospecific antibody binding CD3, as a protein of the present inventioncomprising such characteristics may be used in other formats asdescribed herein, such as in a bispecific or multispecific antibody.

In a particular embodiment, the protein, when present as a monospecificantibody binding CD3, mediates reduced Fc-mediated CD69 expression by atleast 50%, such as at least 60%, at least 70%, at least 80%, at least90%, at least 99%, and 100% when compared to a wild-type protein, whenthe CD69 expression is determined in a peripheral blood mononuclear cell(PBMC)-based functional assay.

In another embodiment, the protein, when present as a monospecificantibody binding CD3, mediates reduced Fc-mediated T-cell activationcompared to a wild-type protein by at least 50%, such as at least 60%,at least 70%, at least 80%, at least 90%, at least 99% and 100%, whenthe T-cell activation is determined by CD69 expression in a peripheralblood mononuclear cells (PBMC)-based functional assay.

In a particular embodiment, the protein, when present as a monospecificantibody binding CD3, mediates reduced CD69 expression when compared toa wild-type protein by at least 50%, such as at least 60%, at least 70%,at least 80%, at least 90%, at least 99%, and 100%, when the CD69expression is measured in a peripheral blood mononuclear cell(PBMC)-based functional assay comprising the steps of (i) incubatingPBMCs with an antibody at 37° C. in a 5% (vol/vol) CO₂ humidifiedincubator for about 16-24 hrs., (ii) washing the cells, (iii) stainingthe cells at 4° C. with a mouse anti-human CD28-PE and mouse-anti-humanCD69-APC antibody, and (iv) determining the CD69 expression on CD28positive cells by flow cytometry.

The term “reduced” as used herein when referring to expression level ofthe T-cell activation marker CD69, refers to a reduction in expressionlevel of CD69 when compared to expression level of CD69 when the T-cellis bound by a wild-type protein provided that both the binding regionsof the protein binds CD3. A protein's ability to reduce expression ofCD69 may be evaluated by a PBMC-based functional assay, as described inExample 3. Thus, in one embodiment, expression of CD69 is measured by amethod comprising the steps of incubating PBMCs with an antibody in therange of 1-1000 ng/mL at 37° C. in a 5% (vol/vol) CO₂ humidifiedincubator for 16-24 hrs., washing the cells, staining the cells at 4° C.with a mouse anti-human CD28-PE and mouse-anti-human CD69-APC antibody,and determining CD69-expression on CD28 positive cells by flowcytometry.

The term “CD69” as used herein, refers to Cluster of Differentiation 69which is a human transmembrane C-Type lectin protein encoded by the CD69gene. Activation of T lymphocytes and natural killer (NK) cells, both invivo and in vitro, induces expression of CD69. CD69 function as a signaltransmitting receptor involved in cellular activation events includingproliferation, functions as a signal-transmitting receptor inlymphocytes, including natural killer cells and platelets, and theinduction of specific genes.

The term “peripheral blood mononuclear cell (PBMC)-based functionalassay” as used herein refers to an assay used for evaluating afunctional feature of the protein of the present invention, such as theability of said protein to affect T-cell proliferation or CD69expression, wherein the only cells present are peripheral bloodmononuclear cells. A PBMC-based functional assay as described in Example3 comprises the steps of (i) incubating PBMCs with an antibody at 37° C.in a 5% (vol/vol) CO₂ humidified incubator for about 16-24 hrs., (ii)washing the cells, (iii) staining the cells at 4° C. with a mouseanti-human CD28-PE and mouse-anti-human CD69-APC antibody, and (iv)determining the CD69 expression on CD28 positive cells by flowcytometry, when CD69 expression is evaluated.

The ability of anti-CD3 antibodies to induce T cell activation, orpotentially agonistic antibodies that can activate T-cells after bindingand cross-linking, is dependent on their FcR binding abilities. Thepresent invention provides a protein, which does not result in CD69expression on T-cells indicating that the protein according to theinvention does not enable Fcγ Receptor binding. The term “Fcγ Receptor”as used herein, refers to a group of Fc Receptors belonging to theimmunoglobulin superfamily and is the most important Fc receptors forinducing phagocytosis of opsonized (coated) microbes. This familyincludes several members, FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB(CD32b), FcγRIIIA (CD16a), FcγRIIIB (CD16b), which differ in theirantibody affinities due to their different molecular structure.

The inventors of the present invention have shown that no or lowexpression of CD69 is observed when a monospecific antibody comprisingthe amino acid substitutions L234F, L235E, and D265A binds to the T-cell(see Example 3 and 11). Thus, in a specific embodiment, the expressionlevel of the T-cell activation marker CD69 is completely reduced.

The term “Fc-mediated T-cell activation” as used herein, refers to anyactivation of the T-cells which are mediated by binding of an antibodyFc region to FcR on FcR-bearing cells. The Fc region refers to a regionof the protein comprising, in the direction from the N- to C-terminal,at least a hinge region, a CH2 domain and a CH3 domain. An Fc region ofan IgG1 antibody can, for example, be generated by digestion of an IgG1antibody with papain.

The term “hrs.” as used herein, refers to the abbreviation of “hours”.In another embodiment, the protein, when present as a monospecificantibody binding CD3, mediates reduced Fc-mediated T-cell proliferationcompared to a wild-type protein by at least 50%, such as at least 60%,at least 70%, at least 80%, at least 90%, at least 99%, and 100%, whenthe T-cell proliferation is measured in a PBMC-based functional assay.

In one embodiment, the protein, when present as a monospecific antibodybinding CD3, mediates reduced Fc-mediated T-cell proliferation by atleast 50%, such as at least 60%, at least 70%, at least 80%, at least90%, at least 99%, and 100%, when the T-cell proliferation is measuredin a PBMC-based functional assay comprising the steps of (i) incubatingPBMCs with an antibody at 37° C. in a 5% (vol/vol) CO₂ humidifiedincubator for three days, (ii) adding an agent capable of incorporationinto the cell DNA, (iii) incubating for 3 to 8 hrs., (iv) pelletingcells, (v) coating cells to an ELISA plate, (vi) incubating with ananti-DNA incorporated agent for 60 to 120 min at room temperature.

In a particular embodiment, the protein, when present as a monospecificantibody binding CD3, mediates reduced Fc-mediated T-cell proliferationby at least 50%, such as at least 60%, at least 70%, at least 80%, atleast 90%, at least 99%, and 100%, when the T-cell proliferation ismeasured in a PBMC-based functional assay comprising the steps of (i)incubating PBMCs with an antibody at 37° C. in a 5% (vol/vol) CO₂humidified incubator for three days, (ii) adding BrdU, (iii) incubatingfor five hrs., (iv) pelleting cells, (v) coating cells to an ELISAplate, (vi) incubating with anti-BrdU-peroxidase for 90 min at roomtemperature, (vii) developing the plate for about 30 min with 1 mg/mL2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), (viii) adding100 μL 2% oxalic acid to stop the reaction, and (xi) measuringabsorbance at 405 nm.

The term “reduced” as used herein when referring to T-cellproliferation, refers to a reduction in the ability of the proteinaccording to the invention to induce proliferation of T-cells whencompared to the induction of proliferation of T-cells bound by awild-type protein provided that both the binding regions of the proteinbinds CD3. The reduction in ability of a protein to induce T-cellproliferation may be evaluated by a PBMC-based functional assay, asdescribed in Example 4. Thus, in one embodiment, T-cell proliferation ismeasured by a method comprising the steps of incubating PBMCs withantibody in the range of 1-1000 ng/mL at 37° C. in a 5% (vol/vol) CO₂humidified incubator for three days, adding a chemical compound, such asBrdU, which is incorporated into the DNA of proliferating cells,incubating for five hrs., pelleting cells, drying cells, optionallystoring the cells at 4° C., coating cells to ELISA plates, incubatingwith anti-BrdU-peroxidase for 90 min at room temperature, developing forabout 30 min with 1 mg/mL 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), adding 100 μL 2% oxalic acidto stop the reaction, and measuring absorbance at 405 nm in a suitablemicroplate reader.

The term “BrdU” as used herein, refers to 5-bromo-2′-deoxyuridine, whichis a homologue to thymidine. When BrdU is added to cell culture for alimited period of time (e.g. 4 hours) it will be incorporated into theDNA of proliferating cells. After fixing the cells, detection ofincorporated BrdU may be performed in an ELISA usinganti-BrdU-peroxidase. BrdU incorporation is therefore a measure forproliferation.

In a specific embodiment, the T-cell proliferation is completelyreduced. Thus, no proliferation of T-cells may be observed, or the levelof proliferation may be equal to the proliferation of T-cells which hasnot been treated with a protein according to the invention or has beentreated with a wild-type protein. The invention provides an antibodycomprising the amino acid substitutions L234F, L235E, and D265A, whichresults in absolutely no proliferation of T-cells, indicating that theprotein according to the invention does not enable Fcγ Receptor binding,as described in Example 4 and 12.

Furthermore, an antibody according to the invention, i.e. an antibodycomprising the amino acid substitutions L234F, L235E, and D265A, hasbeen shown to retain its ability to kill tumor cells. As can be seen inExample 5, the amino acid substitutions according to the presentinvention have little effect on efficacy of the antibodies. Testing aprotein according to the present invention for cytotoxicity efficacy,may be performed in a cytotoxicity assay comprising the steps of (i)putting tumor cells in a cell plate, (ii) adding samples, such asantibody dilutions, in a dose response series, (iii) incubating the cellplate for about 3 hrs., (iv) adding isolated PBCMs from whole blood, (v)incubating plates for three days, (vi) washing plates, (vii) incubatingplates with culture medium containing 10% Alamar Blue for four hrs., and(viii) measuring cell viability.

A monospecific antibody fulfilling the assay conditions herein describedmay form the basis of a bispecific antibody, i.e. in a bispecificantibody wherein one of the binding regions binds CD3 may originate fromany monospecific CD3 antibody tested for the ability of mediatingreduced CD69 expression and/or Fc-mediated T-cell proliferation in thefunctional assays, and fulfilling the requirements, described herein.

The inventors of the present invention have shown, that a bispecificantibody comprising the amino acid substitutions L234F, L235E, andD265A, and which first binding region binds CD3 and which second bindingregion binds a cancer-specific antigen (HER2), that besides the abilityof inducing dose-dependent killing of AU365 cells with at leastcomparable efficacy compared to the wild-type bispecific antibodywithout the non-activating mutation (as described in Example 5 and 13),the bispecific antibody according to the invention also showed cytokinerelease caused by unspecific killing is inhibited, as described inExample 6. It was shown, that incubation of target and effector cells inthe presence of a bispecific antibody according to the invention, didnot lead to any cytokine production caused by unspecific killing (asdescribed in Example 5), whereas the wild-type antibody showed a highercytokine production which is believed to be due to the unspecificactivation of T-cells via cross-linking with other effector cells. Thus,in one embodiment, in the first and second polypeptides the amino acidsin positions corresponding to position L234, L235, and D265, in a humanIgG1 heavy chain are not L, L, and D, said first binding region bindsCD3, and said second binding region binds a tumor-specific target.

The term “cytokine release” as used herein is intended to refer to therelease of cytokines, such as interleukins and interferons, uponactivation of e.g. T-cells. Cytokines are involved in a broad array ofbiological activities including innate immunity, adaptive immunity, andinflammation. Cytokines may activate the cell types from which they havebeen released and thus, stimulating to produce more cytokines.

The ability of a protein according to the invention to induce cytokinerelease may be determined in an assay comprising the steps of (i)incubating supernatant from a cytotoxicity assay as described above forabout 1 to 2 hrs. at room temperature in a plate, (ii) incubatingfurther 1 to 2 hrs. with added solution comprising antibodies againstthe cytokines to be tested, such solution may be a Detection AntibodySolution to the plate, (iii) washing the plate, (iv) adding a conductorto enhance the signal to be read, such as a Read Buffer T, and (v)measuring chemiluminescence.

As described above, complement activation is an effector function whichsome antibodies are able to induce. The first step in the complementcascade is Fc binding of C1q and therefore serves as an indicator forCDC capacity of antibodies. As the present invention relates toinertness of antibodies, complement activation is not wanted andtherefore, deposition of C1q to antibodies was determined in by ELISA asdescribed in Example 7. As determined in said Example, an antibodyaccording to the present invention abrogates C1q binding which suggeststhat the antibodies of the present invention are not capable of inducingCDC.

The term “C1q binding” as used herein is intended to refer to thebinding of C1q to an antibody, when said antibody is bound to itsantigen. The term “bound to its antigen” as used herein refers both tobinding of an antibody to its antigen in vivo and in vitro.

Thus, the ability of a protein according to the present invention of C1qbinding may be determined by ELISA comprising the steps of adding ananti-human C1q and adding an anti-rabbit IgG-Fc-HRP antibody.

Specifically, the ability of a protein according to the presentinvention of C1q binding may be determined by ELISA comprising the stepsof (i) coating an ELISA plate with a dilution series of an protein, (ii)blocking the plate, (iii) adding 3% serum, (iv) incubating the plate for1 hr at 37° C., (v) washing the plate, (vi) adding an anti-human C1q,(vi) incubating the plate for 1 hr at room temperature, (vii) washingthe plate, (viii) adding an anti-rabbit IgG-Fc-HRP antibody, (ix)incubating the plate for 1 hr at room temperature, (x) washing theplate, (xi) developing the plate, and (xii) measuring OD405 nm.

Further analysis of a protein according to the present invention mayinclude determining FcγRI binding as described in Example 8. Asdescribed above, Fcγ Receptors is a group of Fc Receptors, whichcomprise of five different variants of Fcγ Receptors. FcγRI is a highaffinity Fc receptor, which means that the binding between an FcγRI andan Fc region of a protein is strong. If binding to the FcγRI can beinhibited or even abrogated, it is a good indicator of the inertness ofa protein. Thus, evaluating the Fc binding of a protein to FcγRI, isanother way of determining the inertness of said protein. In oneembodiment the protein according to the present invention has completelyabrogated Fc binding to FcγRI. FcγRI binding ability of a proteinaccording to the present invention may be evaluated by flow cytometrycomprising the steps of (i) incubating FcγRI positive cells for approx.30 min at 4° C. with an antibody to be tested; (ii) washing the cells,(iii) staining the cells for approx. 30 min at 4° C. with an anti-humanIgG-PE F(ab′)2 antibody, (iv) washing the cells, and (v) measuring themean fluorescence of the cells.

The term “approx.” as used herein refers to an abbreviation of the term“approximately”.

As described herein an antibody according to the invention, wherein saidfirst and second polypeptides of said antibody comprise the amino acidsubstitutions L234F, L235E, and D265A, and the first binding regionbinds CD3, has proven to completely reduce expression of CD69 andthereby abrogating T-cell activation (Example 3), completely reduceT-cell proliferation (Example 4), completely reduce unspecific killing(Example 5), completely reduce cytokine release when present as amonospecific antibody binding CD3, i.e. the two binding regions of theantibody binds the same target (Example 6), abrogate C1q binding(Example 7) as well as completely reduce FcγRI binding (Example 8) whencompared to a wild-type antibody. Therefore, it is believed that theamino acid positions corresponding to positions L234, L235 and D265 in ahuman IgG1 heavy chain are crucial positions in an antibody in order toprovide an antibody that has the before-mentioned features.

Thus, in one embodiment, in at least one of said first and secondpolypeptides the amino acids corresponding to positions L234 and L235 ina human IgG1 heavy chain are selected from the group consisting of; A,C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V, and W, and the aminoacid corresponding to position D265 is selected from the groupconsisting of; A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, andW.

In one embodiment, in at least one of said first and second polypeptidesthe amino acids in the positions corresponding to positions L234, L235and D265 in a human IgG1 heavy chain are hydrophobic or polar aminoacids.

The term “hydrophobic” as used herein in relation to an amino acidresidue, refers to an amino acid residue selected from the groupconsisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y. Thus, in oneembodiment, in at least one of said first and second polypeptides theamino acid in the position corresponding to position D265 in a humanIgG1 heavy chain is selected from the group of amino acids consistingof; A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group consisting of; A, C, F, G, H, I,M, R, T, V, W, and Y.

The term “polar” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of;C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in at leastone of said first and second polypeptides the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group of amino acids consisting of; C,D, E, H, K, N, Q, R, S, and T, the amino acid in the positioncorresponding to position D265 in a human heavy chain is selected fromthe group consisting of; C, E, H, K, N, Q, R, S, and T.

In a particular embodiment, in at least one of said first and secondpolypeptides the amino acids in the positions corresponding to positionsL234 and L235 in a human IgG1 heavy chain are each selected from thegroup consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W,and Y, and the amino acid in the position corresponding to position D265in a human IgG1 heavy chain is selected from the group consisting of; A,C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

In one embodiment, in both said first and second polypeptides the aminoacids in the positions corresponding to L234, L235, and D265 in a humanIgG1 heavy chain are hydrophobic or polar amino acids.

In one embodiment, in both said first and second polypeptides the aminoacid in the position corresponding to position D265 in a human IgG1heavy chain is selected from the group of amino acids consisting of; A,C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in thepositions corresponding to positions L234 and L235 in a human IgG1 heavychain are each selected from the group consisting of; A, C, F, G, H, I,M, R, T, V, W, and Y.

In one embodiment, in both said first and second polypeptides the aminoacids in the positions corresponding to positions L234 and L235 in ahuman IgG1 heavy chain are each selected from the group of amino acidsconsisting of; C, D, E, H, K, N, Q, R, S, and T, the amino acid in theposition corresponding to position D265 in a human heavy chain isselected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

In a particular embodiment, in both said first and second polypeptidesthe amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain are each selected from the groupconsisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y,and the amino acid in the position corresponding to position D265 in ahuman IgG1 heavy chain is selected from the group consisting of; A, C,E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

In another embodiment, in at least one of said first and secondpolypeptides the amino acids in the positions corresponding to positionsL234, L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged,aromatic or acidic amino acids.

The term “aliphatic uncharged” as used herein in relation to amino acidresidues, refers to any amino acid residue selected from the groupconsisting of: A, G, I, L, and V. Thus, in one embodiment, in at leastone of said first and second polypeptides the amino acid in the positioncorresponding to position D265 in a human IgG1 heavy chain is selectedfrom the group consisting of; A, G, I, L, and V, and the amino acids inthe positions corresponding to positions L234 and L235 in a human IgG1heavy chain are each selected from the group consisting of; A, G, I, andV.

The term “aromatic” as used herein in relation to amino acid residues,refers to any amino acid residue selected from the group consisting of:F, T, and W. Thus, in one embodiment, in at least one of said first andsecond polypeptides the amino acids in the positions corresponding topositions L234, L235 and D265 in a human IgG1 heavy chain are eachselected from the group consisting of; F, T, and W.

The term “acidic” as used herein in relation to amino acid residues,refers to any amino acid residue chosen from the group consisting of: Dand E. Thus, in one embodiment, in at least one of said first and secondpolypeptides the amino acids in the positions corresponding to positionsL234, L235, and D265 in a human IgG1 heavy chain are each selected fromthe group consisting of; D and E.

In a particular embodiment, in at least one of said first and secondpolypeptides the amino acid in the position corresponding to positionD265 in a human IgG1 heavy chain is selected from the group consistingof; A, E, F, G, I, L, T, V, and W, and the amino acids in the positionscorresponding to L234 and L235 are each selected from the groupconsisting of; A, D, E, F, G, I, T, V, and W.

In one embodiment, in both said first and second polypeptides the aminoacids in the positions corresponding to L234, L235, and D265 in a humanIgG1 heavy chain are aliphatic uncharged, aromatic or acidic aminoacids.

In one embodiment, in both said first and second polypeptides the aminoacid in the position corresponding to position D265 in a human IgG1heavy chain is selected from the group consisting of; A, G, I, L, and V,and the amino acids in the positions corresponding to positions L234 andL235 in a human IgG1 heavy chain are each selected from the groupconsisting of; A, G, I, and V.

In one embodiment, in both said first and second polypeptides the aminoacids in the positions corresponding to positions L234, L235, and D265in a human IgG1 heavy chain are each selected from the group consistingof; D and E.

In a particular embodiment, in both said first and second polypeptidesthe amino acid in the position corresponding to position D265 in a humanIgG1 heavy chain is selected from the group consisting of; A, E, F, G,I, L, T, V, and W, and the amino acids in the positions corresponding toL234 and L235 are each selected from the group consisting of; A, D, E,F, G, I, T, V, and W.

In a further embodiment, in at least one of said first and secondpolypeptides, the amino acids corresponding to positions L234, L235 andD265 in a human IgG1 heavy chain are F, E, and A; or A, A, and A,respectively.

In one embodiment, in both said first and second polypeptides the aminoacids in the positions corresponding to position L234, L235, and D265 ina human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively.

In a particular embodiment, in at least one of said first and secondpolypeptides the amino acids corresponding to positions L234, L235 andD265 in a human IgG1 heavy chain are F, E, and A, respectively.

In one embodiment, in both said first and second polypeptides the aminoacids in the positions corresponding to position L234, L235, and D265 ina human IgG1 heavy chain are F, E, and A, respectively.

In one embodiment, in at least one of said first and second polypeptidesthe amino acids corresponding to positions L234, L235, and D265 in ahuman IgG1 heavy chain are A, A, and A, respectively.

In one embodiment, in both said first and second polypeptides the aminoacids in the positions corresponding to positions L234, L235, and D265in a human IgG1 heavy chain are A, A, and A, respectively.

The term “isotype” as used herein refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3, and IgG4) or any allotypes thereof suchas IgG1m(za) and IgG1m(f)) that is encoded by heavy chain constantregion genes. Further, each heavy chain isotype can be combined witheither a kappa (κ) or lambda (λ) light chain or any allotypes thereof.

Thus, in one embodiment, the isotype of the immunoglobulin heavy chainsis selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. Theimmunoglobulin heavy chain may be any allotype within each of theimmunoglobulin classes, such as IgG1m(f) (SEQ ID NO:13). Thus, in oneparticular embodiment, the isotype of the immunoglobulin heavy chainsare an IgG1, or any allotype thereof, such as IgG1m(f).

In another embodiment, at least said first binding region is selectedfrom the group consisting of;

a. a binding region comprising heavy chain variable region sequence asset out in SEQ ID NO:6 and light chain variable region sequence as setout in SEQ ID NO:12;

b. a binding region comprising heavy chain variable region sequence asset out in SEQ ID NO:8 and light chain variable region sequence as setout in SEQ ID NO:12; and

c. a binding region comprising heavy chain variable region sequence asset out in SEQ ID NO:9 and light chain variable region sequence as setout in SEQ ID NO:10.

In one embodiment, both said first and second polypeptides are accordingto any of the embodiments described above.

In one embodiment, the second binding region binds a different targetthan said first binding region. Thus, the protein is a bispecificprotein, such as a bispecific antibody. The term “bispecific protein” or“bispecific antibody” refers to a protein or antibody havingspecificities for two different, typically non-overlapping, epitopes,and comprises two different binding regions. Such epitopes may be on thesame or different targets. If the epitopes are on different targets,such targets may be on the same cell or different cells or cell types.

The term “target” as used herein refers to a molecule to which thebinding region of the protein according to the invention binds. Whenused in the context of the binding of an antibody includes any antigentowards which the raised antibody is directed.

Thus, in one embodiment, the protein is a bispecific antibody.

There is a range of applications, such as receptor inhibition or T-cellrecruitment by bispecific antibodies, in which the Fc binding of the Fcregion of therapeutic antibodies to effector cells or complement is notrequired or even is undesired as it may contribute to unwantedcytotoxicity. Thus, a bispecific antibody which binds with one bindingregion to human CD3 will be able to recruit cytotoxic T-cells. CD3bispecific antibodies with an activating IgG Fc region can induceunwanted agonism in the absence of tumor cells through crosslinking byFcγR-expressing cells, inappropriate activation of FcγR-expressing cellsand subsequent cytokine storm and associated toxic effects, or plateletaggregation. Thus, CD3 bispecific antibodies with a non-activating Fcregion are advantageous to prevent potential unwanted cell activation.

A bispecific antibody according to the present invention is not limitedto any particular bispecific format or method of producing it.

Examples of bispecific antibody molecules which may be used in thepresent invention comprise (i) a single antibody that has two armscomprising different antigen-binding regions, (ii) a single chainantibody that has specificity to two different epitopes, e.g., via twoscFvs linked in tandem by an extra peptide linker; (iii) adual-variable-domain antibody (DVD-Ig), where each light chain and heavychain contains two variable domains in tandem through a short peptidelinkage (Wu et al., Generation and Characterization of a Dual VariableDomain Immunoglobulin (DVD-IgTM) Molecule, In: Antibody Engineering,Springer Berlin Heidelberg (2010)); (iv) a chemically-linked bispecific(Fab′)₂ fragment; (v) a Tandab, which is a fusion of two single chaindiabodies resulting in a tetravalent bispecific antibody that has twobinding sites for each of the target antigens; (vi) a flexibody, whichis a combination of scFvs with a diabody resulting in a multivalentmolecule; (vii) a so called “dock and lock” molecule, based on the“dimerization and docking domain” in Protein Kinase A, which, whenapplied to Fabs, can yield a trivalent bispecific binding proteinconsisting of two identical Fab fragments linked to a different Fabfragment; (viii) a so-called Scorpion molecule, comprising, e.g., twoscFvs fused to both termini of a human Fab-arm; and (ix) a diabody.

In one embodiment, the bispecific antibody of the present invention is adiabody, a cross-body, or a bispecific antibody obtained via acontrolled Fab arm exchange (such as described in WO 11/131746) as thosedescribed in the present invention.

Examples of different classes of bispecific antibodies include but arenot limited to (i) IgG-like molecules with complementary CH3 domains toforce heterodimerization; (ii) recombinant IgG-like dual targetingmolecules, wherein the two sides of the molecule each contain the Fabfragment or part of the Fab fragment of at least two differentantibodies; (iii) IgG fusion molecules, wherein full length IgGantibodies are fused to extra Fab fragment or parts of Fab fragment;(iv) Fc fusion molecules, wherein single chain Fv molecules orstabilized diabodies are fused to heavy-chain constant-domains,Fc-regions or parts thereof; (v) Fab fusion molecules, wherein differentFab-fragments are fused together, fused to heavy-chain constant-domains,Fc-regions or parts thereof; and (vi) ScFv- and diabody-based and heavychain antibodies (e.g., domain antibodies, nanobodies) wherein differentsingle chain Fv molecules or different diabodies or differentheavy-chain antibodies (e.g. domain antibodies, nanobodies) are fused toeach other or to another protein or carrier molecule fused toheavy-chain constant-domains, Fc-regions or parts thereof.

Examples of IgG-like molecules with complementary CH3 domains moleculesinclude but are not limited to the Triomab/Quadroma (TrionPharma/Fresenius Biotech; Roche, WO2011069104), the Knobs-into-Holes(Genentech, WO9850431), CrossMAbs (Roche, WO2011117329) and theelectrostatically-matched (Amgen, EP1870459 and WO2009089004; Chugai,US201000155133; Oncomed, WO2010129304), the LUZ-Y (Genentech), DIG-bodyand PIG-body (Pharmabcine), the Strand Exchange Engineered Domain body(SEEDbody)(EMD Serono, WO2007110205), the Biclonics (Merus), FcAAdp(Regeneron, WO201015792), bispecific IgG1 and IgG2 (Pfizer/Rinat,WO11143545), Azymetric scaffold (Zymeworks/Merck, WO2012058768), mAb-Fv(Xencor, WO2011028952), bivalent bispecific antibodies (Roche) and theDuoBody (Genmab A/S, WO2011131746).

Examples of recombinant IgG-like dual targeting molecules include butare not limited to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-oneAntibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb²(F-Star, WO2008003116), Zybodies (Zyngenia), approaches with commonlight chain (Crucell/Merus, U.S. Pat. No. 7,262,028), KABodies(NovImmune) and CovX-body (CovX/Pfizer).

Examples of IgG fusion molecules include but are not limited to DualVariable Domain (DVD)-Ig (Abbott, U.S. Pat. No. 7,612,181), Dual domaindouble head antibodies (Unilever; Sanofi Aventis, WO20100226923),IgG-like Bispecific (ImClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb(Zymogenetics), HERCULES (Biogen Idec, U.S. Ser. No. 00/795,1918), scFvfusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc, CN102250246) and TvAb (Roche, WO2012025525, WO2012025530).

Examples of Fc fusion molecules include but are not limited to ScFv/FcFusions (Academic Institution), SCORPION (Emergent BioSolutions/Trubion,Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART)(MacroGenics, WO2008157379, WO2010/080538) and Dual(ScFv)₂-Fab (NationalResearch Center for Antibody Medicine—China).

Examples of Fab fusion bispecific antibodies include but are not limitedto F(ab)₂ (Medarex/AMGEN), Dual-Action or Bis-Fab (Genentech),Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol) andFab-Fv (UCB-Celltech).

Examples of ScFv-, diabody-based and domain antibodies include but arenot limited to Bispecific T Cell Engager (BITE) (Micromet, TandemDiabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART)(MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies(AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack) andCOMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dualtargeting heavy chain only domain antibodies.

It is further contemplated that any monospecific antibody fulfilling theassay conditions herein described may form the basis of a bispecificantibody, i.e. in a bispecific antibody wherein one of the bindingregions binds CD3 may originate from any monospecific CD3 antibodytested in the functional assays and fulfilling the requirements statedherein. Such bispecific antibody may be provided by the methodsdescribed in WO 2011/131746, which is hereby incorporated by reference.

Thus, in a particular embodiment, in said first polypeptide at least oneof the amino acids in the positions corresponding to a position selectedfrom the group consisting of T366, L368, K370, D399, F405, Y407, andK409 in a human IgG1 heavy chain has been substituted, and in saidsecond polypeptide at least one of the amino acids in the positionscorresponding to a position selected from the group consisting of; T366,L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain hasbeen substituted, and wherein said substitutions of said first and saidsecond polypeptides are not in the same positions. In this context theterm “substituted”, refers to the amino acid in a specific amino acidposition which has been substituted with another naturally ornon-naturally occurring amino acid. Thus, a “substituted” amino acid ina position corresponding to the position in a human IgG1 heavy chainmeans the amino acid at the particular positions is different from thenaturally occurring amino acid in an IgG1 heavy chain.

In one embodiment, in said first polypeptide the amino acid in theposition corresponding to K409 in a human IgG1 heavy chain is not K, Lor M, and the amino acid in the position corresponding to F405 in ahuman IgG1 heavy chain is F, and in said second polypeptide at least oneof the amino acids in the positions corresponding to a position selectedfrom the group consisting of; T366, L368, K370, D399, F405, and Y407 ina human IgG1 heavy chain has been substituted.

In one embodiment, in said first polypeptide the amino acid in theposition corresponding to K409 in a human IgG1 heavy chain is not K, Lor M, and the amino acid in the position corresponding to F405 in ahuman IgG1 heavy chain is F, and in said second polypeptide the aminoacid in the position corresponding to F405 in a human IgG1 heavy chainis not F and the amino acid in the position corresponding to K409 in ahuman IgG1 heavy chain is K.

In one embodiment, in said first polypeptide, the amino acid in theposition corresponding to F405 in a human IgG1 heavy chain is not F, R,and G, and the amino acid in the position corresponding to K409 in ahuman IgG1 heavy chain is K, and in said second polypeptide the aminoacids in the positions corresponding to a position selected form thegroup consisting of; T366, L368, K370, D399, Y407, and K409 in a humanIgG1 heavy chain has been substituted.

In one embodiment, in said first polypeptide, the amino acid in theposition corresponding to K409 in a human IgG1 heavy chain is not K, Lor M, and the amino acid in the position corresponding to F405 in ahuman IgG1 heavy chain is F, and the amino acid in positioncorresponding to F405 in a human IgG1 heavy chain is not F, and theamino acid in the position corresponding to K409 in a human IgG1 heavychain is K.

In a further embodiment, the amino acid in the position corresponding toF405 in a human IgG1 heavy chain is L in said first polypeptide, and theamino acid in the position corresponding to K409 in a human IgG1 heavychain is R in said second polypeptide, or vice versa.

In one embodiment, the targets are present on different cells. In aparticular embodiment, the first binding region binds CD3 present on aT-cell and the second binding region binds a tumor-specific targetpresent on a cancer cell. Thereby, the bispecific protein, such as abispecific antibody, binds two different cell types. When both celltypes are engaged, activation of the T-cells, particularly the cytotoxicT-cells (CD8+ T-cells), will be triggered by the specific interactionbetween the T-cells and the cancer/tumor cells. Thus, the proteinaccording to the present invention provides an attractive way ofactivating T-cells and killing cancer cells.

Thus, in a particular embodiment, the protein is a bispecific antibody,both said first and second polypeptide the amino acids in the positionscorresponding to L234, L235 and D265 in a human IgG1 heavy chain are F,E, and A, respectively, said first binding region binds CD3, and saidsecond binding region binds a cancer-specific target.

In one embodiment, the protein according to any aspects or embodimentsherein described is an antibody. In one embodiment, the protein is abispecific antibody. In one embodiment, the antibody is a full-lengthantibody or a human antibody. In one embodiment, the antibody is a humanIgG1 antibody.

Furthermore, it is contemplated that a wild-type protein according toany aspect or embodiment herein described may also be a parent protein.Thus, the present invention also relates to a variant of such a parentprotein. Therefore, it is contemplated that any protein according to theinvention may also be regarded as a variant of a parent protein obtainedby modifying the parent protein.

The protein according to the invention may be prepared by a methodcomprising introducing into the first and/or second polypeptides of awild-type protein, amino acid substitutions in the positionscorresponding to L234, L235 and D265 in a human IgG1 heavy chain.

A variant according to the invention may be prepared by a methodcomprising introducing into the first and/or second polypeptides of aparent protein, amino acid substitutions in the positions correspondingto L234, L235, and D265 in a human IgG1 heavy chain.

Methods of preparing an antibody are well-known to the skilled person.However, a non-limiting example of preparing an antibody according tothe invention may be by a method comprising immunizing a non-humananimal, e.g. a mouse, obtaining the antibodies from the non-humananimal, introducing amino acid mutations in the Fc region according tothe invention by recombinant techniques, expressing the nucleic acidsobtained by the recombinant techniques in a suitable expression system,and purifying the expressed antibodies.

As described herein, the present invention relates in one embodiment toan antibody wherein in least one of the two heavy chains of animmunoglobulin the amino acids in the positions corresponding topositions L234, L235, and D265 in a human IGG1 heavy chain, are not L,L, and D, respectively. Thus, the protein of the present invention maybe prepared by introducing mutations into said positions of an antibody.An antibody into which said mutations are introduced may be regarded asa “parent antibody”. The “parent” antibodies, which may be wild-typeantibodies, to be used as starting material of the present inventionbefore modification may e.g. be produced by the hybridoma method firstdescribed by Kohler et al., Nature 256, 495 (1975), or may be producedby recombinant DNA methods. Monoclonal antibodies may also be isolatedfrom phage antibody libraries using the techniques described in, forexample, Clackson et al., Nature 352, 624 628 (1991) and Marks et al.,J. Mol. Biol. 222, 581 597 (1991). Monoclonal antibodies may be obtainedfrom any suitable source. Thus, for example, monoclonal antibodies maybe obtained from hybridomas prepared from murine splenic B cellsobtained from mice immunized with an antigen of interest, for instancein form of cells expressing the antigen on the surface, or a nucleicacid encoding an antigen of interest. Monoclonal antibodies may also beobtained from hybridomas derived from antibody-expressing cells ofimmunized humans or non-human mammals such as rabbits, rats, dogs,primates, etc.

The parent antibodies may be e.g. chimeric or humanized antibodies. Inanother embodiment, the antibody is a human antibody. Human monoclonalantibodies may be generated using transgenic or transchromosomal mice,e.g. HuMAb mice, carrying parts of the human immune system rather thanthe mouse system. The HuMAb mouse contains a human immunoglobulin geneminilocus that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature368, 856 859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar,D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci 764 536 546 (1995)). The preparation ofHuMAb mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287 6295 (1992), Chen, J. et al., International Immunology5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994),Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845 851 (1996). See also U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397,5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according towell-known techniques.

Further, human antibodies of the present invention or antibodies of thepresent invention from other species may be identified throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, mammalian display, yeast displayand other techniques known in the art, and the resulting molecules maybe subjected to additional maturation, such as affinity maturation, assuch techniques are well known in the art.

The parent antibody is not limited to antibodies which have a natural,e.g. a human Fc region but it may also be an antibody having othermutations than those of the present invention, such as e.g. mutationsthat affect glycosylation or enables the antibody to be a bispecificantibody. By the term “natural antibody” is meant any antibody whichdoes not comprise any genetically introduced mutations. An antibodywhich comprises naturally occurred modifications, e.g. differentallotypes, is thus to be understood as a “natural antibody” in the senseof the present invention, and can thereby be understood as a parentantibody. Such antibodies may serve as a template for the one or moremutations according to the present invention, and thereby providing thevariant antibodies of the invention. An example of a parent antibodycomprising other mutations than those of the present invention is thebispecific antibody as described in WO2011/131746 (Genmab) or othermutations related to any bispecific antibody described herein.

The parent antibody may bind any target.

Monoclonal antibodies for use in the present invention, may be produced,e.g., by the hybridoma method first described by Kohler et al., Nature256, 495 (1975), or may be produced by recombinant DNA methods.Monoclonal antibodies may also be isolated from phage antibody librariesusing the techniques described in, for example, Clackson et al., Nature352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991).Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B cells obtained from mice immunized withan antigen of interest, for instance in form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest. Monoclonal antibodies may also be obtained from hybridomasderived from antibody-expressing cells of immunized humans or non-humanmammals such as rats, dogs, primates, etc.

In one embodiment, the antibody is a human antibody. Human monoclonalantibodies directed against any antigen may be generated usingtransgenic or transchromosomal mice carrying parts of the human immunesystem rather than the mouse system. Such transgenic andtranschromosomic mice include mice referred to herein as HuMAb® mice andKM mice, respectively, and are collectively referred to herein as“transgenic mice”.

The HuMAb® mouse contains a human immunoglobulin gene miniloci thatencodes unrearranged human heavy (μ and γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature368, 856-859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar,D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation ofHuMAb® mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287-6295 (1992), Chen, J. et al., International Immunology5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994),Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397,5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in theirendogenous light chain (kappa) genes (as described in Chen et al., EMBOJ. 12, 821-830 (1993)), a CMD disruption in their endogenous heavy chaingenes (as described in Example 1 of WO 01/14424), and a KCo5 human kappalight chain transgene (as described in Fishwild et al., NatureBiotechnology 14, 845-851 (1996)). Additionally, the Hco7 mice have aHCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429), the HCo12 mice have a HCo12 human heavy chain transgene (asdescribed in Example 2 of WO 01/14424), the HCo17 mice have a HCo17human heavy chain transgene (as described in Example 2 of WO 01/09187)and the HCo20 mice have a HCo20 human heavy chain transgene. Theresulting mice express human immunoglobulin heavy and kappa light chaintransgenes in a background homozygous for disruption of the endogenousmouse heavy and kappa light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478. HCo12-Balb/C mice can be generated by crossing HCo12 toKCo5P/KKBalb) as described in WO/2009/097006.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according towell-known techniques.

Further, any antigen-binding regions may be obtained from humanantibodies or antibodies from other species identified throughdisplay-type technologies, including, without limitation, phage display,retroviral display, ribosomal display, and other techniques, usingtechniques well known in the art and the resulting molecules may besubjected to additional maturation, such as affinity maturation, as suchtechniques are well known in the art (see for instance Hoogenboom etal., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al.,Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNASUSA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992),Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. AcidsResearch 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130,43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S.Pat. No. 5,733,743). If display technologies are utilized to produceantibodies that are not human, such antibodies may be humanized.

Systems for expression of the protein and variant according to theinvention are well-known in the art for the skilled person and includebut are not limited to those described herein.

In one aspect, the invention provides a composition comprising theprotein or variant according to any aspects and embodiments hereindescribed.

Nucleic Acids and Expression Constructs

In a further aspect, the present invention relates to a nucleic acidencoding a first or second polypeptide according to the presentinvention, wherein the amino acids in the position corresponding toL234, L235 and D265 in a human IgG1 heavy chain, are not L, L, and D. Itis further contemplated that the nucleic acid encoding a first or secondpolypeptide according to the invention comprises the amino acidsubstitutions in the specific amino acid positions herein described.Thus, in one embodiment, the nucleic acid encodes a first or secondpolypeptide having the sequence according to SEQ ID NO:20. In the aminoacid sequence as set out in SEQ ID NO:20 the specific three amino acidsubstitutions L234F, L235E and D265A have been indicated by bold andunderlined letters.

In another aspect, the invention relates to nucleic acids encoding asequence of a human, humanized or chimeric CD3 antibody for use in theinvention, to expression vectors encoding the sequences of such anantibody, to host cells comprising such expression vectors, tohybridomas which produce such antibodies, and to methods of producingsuch an antibody by culturing such host cells or hybridomas underappropriate conditions whereby the antibody is produced and, optionally,retrieved. Humanized CD3 antibodies may also be denoted as “huCD3”.

In one embodiment, the invention provides an expression vectorcomprising a nucleotide sequence encoding the amino acid sequenceaccording to SEQ ID NOs.:21 or 28.

In one embodiment, the invention provides an expression vectorcomprising a nucleotide sequence encoding one or more amino acidsequences selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9,10, 11, and 12, or any combination thereof. In another embodiment, theexpression vector comprises a nucleotide sequence encoding any one ormore of the VH CDR3 amino acid sequences selected from SEQ ID NOs: 3 and15. In another embodiment, the expression vector comprises a nucleotidesequence encoding a VH amino acid sequence selected from SEQ ID NOs: 6,7, 8, and 9. In another embodiment, the expression vector comprises anucleotide sequence encoding a VL amino acid sequence selected from SEQID NOs: 10, 11, and 12. In another embodiment, the expression vectorcomprises a nucleotide sequence encoding the constant region of a humanantibody light chain, of a human antibody heavy chain, or both. Inanother embodiment, the expression vector comprising a nucleotidesequence encoding the constant region of a human antibody heavy chain ofSEQ ID NOs:13.

In a particular embodiment, the expression vector comprises a nucleotidesequence encoding a variant of one or more of the above amino acidsequences, said variant having at most 25 amino acid modifications, suchas at most 20, such as at most 15, 14, 13, 12, or 11 amino acidmodifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidmodifications, such as deletions or insertions, preferablysubstitutions, such as conservative substitutions, or at least 80%identity to any of said sequences, such as at least 85% identity or 90%identity or 95% identity, such as 96% identity or 97% identity or 98%identity or 99% identity to any of the afore-mentioned amino acidsequences.

An expression vector in the context of the present invention may be anysuitable vector, including chromosomal, non-chromosomal, and syntheticnucleic acid vectors (a nucleic acid sequence comprising a suitable setof expression control elements). Examples of such vectors includederivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeastplasmids, vectors derived from combinations of plasmids and phage DNA,and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ahumanized CD3 antibody-encoding nucleic acid is comprised in a naked DNAor RNA vector, including, for example, a linear expression element (asdescribed in for instance Sykes and Johnston, Nat Biotech 17, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in for instance Schakowski et al., Mol Ther 3,793-800 (2001)), or as a precipitated nucleic acid vector construct,such as a CaPO₄ ⁻-precipitated construct (as described in for instanceWO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigleret al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic CellGenetics 7, 603 (1981)). Such nucleic acid vectors and the usage thereofare well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and5,973,972).

In one embodiment, the vector is suitable for expression of thehumanized CD3 antibody, the first and the second polypeptides in abacterial cell. Examples of such vectors include expression vectors suchas BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J BiolChem 264, 5503-5509 (1989)), pET vectors (Novagen, Madison Wis.) and thelike.

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), and Grant et al., Methods in Enzymol 153,516-544 (1987)).

A nucleic acid and/or vector may also comprise a nucleic acid sequenceencoding a secretion/localization sequence, which can target apolypeptide, such as a nascent polypeptide chain, to the periplasmicspace or into cell culture media. Such sequences are known in the art,and include secretion leader or signal peptides, organelle-targetingsequences (e.g., nuclear localization sequences, ER retention signals,mitochondrial transit sequences, chloroplast transit sequences),membrane localization/anchor sequences (e.g., stop transfer sequences,GPI anchor sequences), and the like.

In an expression vector of the invention, CD3 antibody-encoding nucleicacids and the first and the second polypeptides nucleic acids maycomprise or be associated with any suitable promoter, enhancer, andother expression-facilitating elements. Examples of such elementsinclude strong expression promoters (e.g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In one embodiment, the CD3 antibody-encoding expression vector and thefirst and the second polypeptides expression vector is positioned inand/or delivered to the host cell or host animal via a viral vector.

Such expression vectors may be used for recombinant production of CD3antibodies and the first and the second polypeptides.

In one aspect, the CD3 antibodies and the first and the secondpolypeptides of any aspect or embodiment described herein are providedby use of recombinant eukaryotic or prokaryotic host cell which producesthe antibody. Accordingly, the invention provides a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces a CD3 antibody, the first and the second polypeptides, orimmunoglobulin as defined herein. Examples of host cells include yeast,bacterial and mammalian cells, such as CHO or HEK-293 cells. Forexample, in one embodiment, the host cell comprises a nucleic acidstably integrated into the cellular genome that comprises a sequencecoding for expression of a CD3 antibody described herein. In oneembodiment, the host cell comprises a nucleic acid stably integratedinto the cellular genome that comprise a sequence coding for expressionof a first or a second polypeptide described herein. In anotherembodiment, the host cell comprises a non-integrated nucleic acid, suchas a plasmid, cosmid, phagemid, or linear expression element, whichcomprises a sequence coding for expression of a CD3 antibody, a first ora second polypeptide described herein.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which an expression vectorhas been introduced. It should be understood that such terms areintended to refer not only to the particular subject cell, but also tothe progeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, transfectomas,such as CHO cells, HEK-293 cells, PER.C6, NSO cells, and lymphocyticcells, and prokaryotic cells such as E. coli and other eukaryotic hostssuch as plant cells and fungi.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cells expressing the antibody or a target antigen, such as CHOcells, PER.C6, NSO cells, HEK-293 cells, plant cells, or fungi,including yeast cells.

In a further aspect, the invention relates to a method for producing anantibody of the invention, said method comprising the steps of

a) culturing a hybridoma or a host cell of the invention as describedherein above, and

b) retrieving and/or purifying the antibody of the invention from theculture media.

In a further aspect, the nucleotide sequence encoding a sequence of anantibody further encodes a second moiety, such as a therapeuticpolypeptide. Exemplary therapeutic polypeptides are described elsewhereherein. In one embodiment, the invention relates to a method forproducing an antibody fusion protein, said method comprising the stepsof

a) culturing a host cell comprising an expression vector comprising sucha nucleotide sequence, and

b) retrieving and/or purifying the antibody fusion protein from theculture media.

Pharmaceutical Compositions

In one aspect, the invention provides a pharmaceutical compositioncomprising a protein, such as an antibody, as defined in any of theaspects and embodiments herein described, and a pharmaceuticallyacceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 19thEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the protein,variant or antibody of the present invention and the chosen mode ofadministration. Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen compound or pharmaceutical composition of the present invention(e.g., less than a substantial impact (10% or less relative inhibition,5% or less relative inhibition, etc.)) on antigen binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e. g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the amide thereof, the route of administration,the time of administration, the rate of excretion of the particularcompound being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompositions employed, the age, sex, weight, condition, general healthand prior medical history of the patient being treated, and like factorswell known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a protein, variant orantibody of the present invention in vivo and in vitro are well known inthe art and may be selected by those of ordinary skill in the art.

In one embodiment, a pharmaceutical composition of the present inventionis administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intra-arterial, intrathecal, intracapsular,intra-orbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption-delaying agents,and the like that are physiologically compatible with a protein, variantor antibody of the present invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated. When referring to the “activecompound” it is contemplated to also refer to the protein, the antibody,the variant of a parent protein or patent antibody according to thepresent invention.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Theprotein, variant and antibody of the present invention may be preparedwith carriers that will protect the compound against rapid release, suchas a controlled release formulation, including implants, transdermalpatches, and micro-encapsulated delivery systems. Such carriers mayinclude gelatin, glyceryl monostearate, glyceryl distearate,biodegradable, biocompatible polymers such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, poly-orthoesters, andpolylactic acid alone or with a wax, or other materials well known inthe art. Methods for the preparation of such formulations are generallyknown to those skilled in the art. See e.g., Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,New York, 1978.

In one embodiment, the proteins, antibodies, or variants of a parentprotein or parent antibody of the present invention may be formulated toensure proper distribution in vivo. Pharmaceutically acceptable carriersfor parenteral administration include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated. Other active or therapeuticcompounds may also be incorporated into the compositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or a non-aqueous solvent or dispersion medium containingfor instance water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixturesthereof, vegetable oils, such as olive oil, and injectable organicesters, such as ethyl oleate. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the active compound in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum-drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Therapeutic Applications

In another aspect, the present invention relates to a protein, e.g.antibody, or pharmaceutical composition of the invention as defined inany aspect or embodiment herein described, for use as a medicament.

In another aspect, the present invention relates to a protein, variant,antibody, or pharmaceutical composition of the invention as defined inany aspect or embodiment herein described, for use in the treatment of adisease.

The protein, variant, antibody, or pharmaceutical composition of theinvention can be used as in a treatment wherein the effector mechanismsof cytotoxic T-cells are desired. For example, the protein, variant, orantibody may be administered to cells in culture, e.g., in vitro or exvivo, or to human subjects, e.g. in vivo, to treat or prevent disorderssuch as cancer, inflammatory or autoimmune disorders. As used herein,the term “subject” is typically a human which respond to the protein,variant, antibody, or pharmaceutical composition. Subjects may forinstance include human patients having disorders that may be correctedor ameliorated by modulating a target function or by leading to killingof the cell, directly or indirectly.

In another aspect, the present invention provides methods for treatingor preventing a disorder, such as cancer, wherein recruitment of T-cellswould contribute to the treatment or prevention, which method comprisesadministration of a therapeutically effective amount of a protein,variant, antibody, or pharmaceutical composition of the presentinvention to a subject in need thereof. The method typically involvesadministering to a subject a protein, variant, or antibody in an amounteffective to treat or prevent the disorder.

In one particular aspect, the present invention relates to a method oftreatment of cancer comprising administering the protein, variant,antibody, or pharmaceutical composition of the invention as defined inany aspect and embodiments herein described.

In another aspect, the present invention relates to the use or themethod of the invention as defined in any aspect and embodiments hereindescribed wherein the disease is cancer, inflammatory or autoimmunediseases.

Cells overexpressing tumor-specific targets are particularly goodtargets for the protein, variant or antibody of the invention, sincerecruitment of T-cells by one of the two binding regions of the protein,variant, or antibody will trigger a cytotoxic activity of the T-cells.This mechanism is normally difficult to obtain, as the triggering of acytotoxic activity does not work properly in elimination of cancercells.

The efficient dosages and dosage regimens for the protein, variant, orantibody depend on the disease or condition to be treated and may bedetermined by the persons skilled in the art.

A physician having ordinary skill in the art may readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician could start doses of the protein,variant, or antibody employed in the pharmaceutical composition atlevels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. In general, a suitable dose of a composition of thepresent invention will be that amount of the protein, variant, orantibody which is the lowest dose effective to produce a therapeuticeffect according to a particular dosage regimen. Such an effective dosewill generally depend upon the factors described above.

For example, an “effective amount” for therapeutic use may be measuredby its ability to stabilize the progression of disease. The ability of acompound to inhibit cancer may, for example, be evaluated in an animalmodel system predictive of efficacy in human tumors. Alternatively, thisproperty of a composition may be evaluated by examining the ability ofthe protein, variant, or antibody to inhibit cell growth or to inducecytotoxicity by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound, i.e. atherapeutic protein, variant, antibody, or pharmaceutical compositionaccording to the invention, may decrease tumor size, or otherwiseameliorate symptoms in a subject. One of ordinary skill in the art wouldbe able to determine such amounts based on such factors as the subject'ssize, the severity of the subject's symptoms, and the particularcomposition or route of administration selected.

An exemplary, non-limiting range for a therapeutically effective amountof a protein, variant or antibody of the invention is about 0.001-10mg/kg, such as about 0.001-5 mg/kg, for example about 0.001-2 mg/kg,such as about 0.001-1 mg/kg, for instance about 0.001, about 0.01, about0.1, about 1 or about 10 mg/kg.

Administration may e.g. be intravenous, intramuscular, intraperitoneal,or subcutaneous, and for instance administered proximal to the site ofthe target.

Dosage regimens in the above methods of treatment and uses are adjustedto provide the optimum desired response (e.g., a therapeutic response).For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation.

In one embodiment, the efficacy of the treatment is monitored during thetherapy, e.g. at predefined points in time.

If desired, an effective daily dose of a pharmaceutical composition maybe administered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In another embodiment, the protein,variant, antibody, or pharmaceutical composition is administered by slowcontinuous infusion over a long period, such as more than 24 hours, inorder to minimize any unwanted side effects.

While it is possible for a protein, variant, or antibody of the presentinvention to be administered alone, it is preferable to administer theprotein, variant, or antibody as a pharmaceutical composition asdescribed above.

An effective dose of a protein, variant, or antibody of the inventionmay also be administered using a weekly, biweekly or triweekly dosingperiod. The dosing period may be restricted to, e.g., 8 weeks, 12 weeksor until clinical progression has been established.

In one embodiment, the protein, antibody or variant may be administeredby infusion in a weekly dosage of calculated by mg/m². Such dosages can,for example, be based on the mg/kg dosages provided above according tothe following: dose (mg/kg)×70: 1.8. Such administration may berepeated, e.g., 1 to 8 times, such as 3 to 5 times. The administrationmay be performed by continuous infusion over a period of from 2 to 24hours, such as of from 2 to 12 hours. In one embodiment, the protein,antibody or variant may be administered by slow continuous infusion overa long period, such as more than 24 hours, in order to reduce toxic sideeffects.

In one embodiment, the protein, antibody or variant may be administeredin a weekly dosage of calculated as a fixed dose for up to 8 times, suchas from 4 to 6 times when given once a week. Such regimen may berepeated one or more times as necessary, for example, after 6 months or12 months. Such fixed dosages can, for example, be based on the mg/kgdosages provided above, with a body weight estimate of 70 kg. The dosagemay be determined or adjusted by measuring the amount of protein,antibody or variant of the present invention in the blood uponadministration by for instance taking out a biological sample and usinganti-idiotypic antibodies which target the binding region of theproteins, antibodies or variants of the present invention.

In one embodiment, the protein, antibody or variant may be administeredby maintenance therapy, such as, e.g., once a week for a period of 6months or more.

A protein, antibody or variant may also be administered prophylacticallyin order to reduce the risk of developing cancer, delay the onset of theoccurrence of an event in cancer progression, and/or reduce the risk ofrecurrence when a cancer is in remission.

Parenteral compositions may be formulated in dosage unit form for easeof administration and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages forthe subjects to be treated; each unit contains a predetermined quantityof active compound calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

A protein, variant, antibody, or antibody may also be administeredprophylactically in order to reduce the risk of developing cancer, delaythe onset of the occurrence of an event in cancer progression, and/orreduce the risk of recurrence when a cancer is in remission. This may beespecially useful in patients wherein it is difficult to locate a tumorthat is known to be present due to other biological factors.

Diagnostic Applications

The non-activating protein of the invention may also be used fordiagnostic purposes, using a composition comprising a protein asdescribed herein. Accordingly, the invention provides diagnostic methodsand compositions using the proteins described herein. Such methods andcompositions can be used for purely diagnostic purposes, such asdetecting or identifying a disease, as well as for monitoring of theprogress of therapeutic treatments, monitoring disease progression,assessing status after treatment, monitoring for recurrence of disease,evaluating risk of developing a disease, and the like.

In one aspect, the protein of the present invention are used ex vivo,such as in diagnosing a disease in which cells expressing a specifictarget of interest and to which the protein binds, are indicative ofdisease or involved in the pathogenesis, by detecting levels of thetarget or levels of cells which express the target of interest on theircell surface in a sample taken from a patient. This may be achieved, forexample, by contacting the sample to be tested, optionally along with acontrol sample, with the protein according to the invention underconditions that allow for binding of the protein to the target. Complexformation can then be detected (e.g., using an ELISA). When using acontrol sample along with the test sample, the level of protein orprotein-target complex is analyzed in both samples and a statisticallysignificant higher level of protein or protein-target complex in thetest sample indicates a higher level of the target in the test samplecompared with the control sample.

Examples of conventional immunoassays in which proteins of the presentinvention can be used include, without limitation, ELISA, RIA, FACSassays, plasmon resonance assays, chromatographic assays, tissueimmunohistochemistry, Western blot, and/or immunoprecipitation.

In one embodiment, the invention relates to a method for detecting thepresence of a target, or a cell expressing the target, in a samplecomprising:

-   -   contacting the sample with a protein of the invention under        conditions that allow for binding of the protein to the target        in the sample; and    -   analyzing whether a complex has been formed. Typically, the        sample is a biological sample.

In one embodiment, the sample is a tissue sample known or suspected ofcontaining a specific target and/or cells expressing the target. Forexample, in situ detection of the target expression may be accomplishedby removing a histological specimen from a patient, and providing theprotein of the present invention to such a specimen. The protein may beprovided by applying or by overlaying the protein to the specimen, whichis then detected using suitable means. It is then possible to determinenot only the presence of the target or target-expressing cells, but alsothe distribution of the target or target-expressing cells in theexamined tissue (e.g., in the context of assessing the spread of cancercells). Using the present invention, those of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) may be modified in order to achieve suchin situ detection.

In the above assays, the protein can be labeled with a detectablesubstance to allow bound protein to be detected. Alternatively, bound(primary) specific protein may be detected by an antibody which islabeled with a detectable substance and which binds to the primaryspecific protein.

The level of target in a sample can also be estimated by a competitionimmunoassay utilizing target standards labeled with a detectablesubstance and an unlabeled target-specific protein. In this type ofassay, the biological sample, the labeled target standard(s) and thetarget-specific protein are combined, and the amount of labeled targetstandard bound to the unlabeled target-specific protein is determined.The amount of target in the biological sample is inversely proportionalto the amount of labeled target standard bound to the target-specificprotein.

Suitable labels for the target-specific protein, secondary antibodyand/or target standard used in in vitro diagnostic techniques include,without limitation, various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,(3-galactosidase, and acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin;an example of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S, and ³H.

In one aspect, the target-specific proteins of the invention are used inthe in vivo imaging of target-expressing tissues such as tumors. For invivo methods, antibody fragments such as, e.g., (Fab′)₂, Fab and Fab′fragments, are particularly advantageous because of their rapiddistribution kinetics.

In vivo imaging can be performed by any suitable technique. For example,a target-specific protein (such as, e.g., an antibody or a fragment)labeled with ⁹⁹Tc, ¹³¹I, ¹¹¹In or other gamma-ray emitting isotope maybe used to image target-specific protein accumulation or distribution intarget-expressing tissues such as tumors with a gamma scintillationcamera (e.g., an Elscint Apex 409ECT device), typically usinglow-energy, high resolution collimator or a low-energy all-purposecollimator. Alternatively, labeling with ⁸⁹Zr, ⁷⁶Br, ¹⁸F or otherpositron-emitting radionuclide may be used to image target-specificprotein, antibody, or antibody fragment distribution in tumors usingpositron emission tomography (PET). The images obtained by the use ofsuch techniques may be used to assess biodistribution of target in apatient, mammal, or tissue, for example in the context of using targetas a biomarker for the presence of cancer/tumor cells. Variations onthis technique may include the use of magnetic resonance imaging (MRI)to improve imaging over gamma camera techniques. Conventionalimmunoscintigraphy methods and principles are described in, e.g.,Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging AndTherapy (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in Remington's Pharmaceutical Sciences, 18th Edition,Gennaro et al., (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in Biotechnology AndPharmacy 227-49, Pezzuto et al., (eds.) (Chapman & Hall 1993). Moreover,such images may also, or alternatively, serve as the basis for surgicaltechniques to remove tumors. Furthermore, such in vivo imagingtechniques may allow for the identification and localization of a tumorin a situation where a patient is identified as having a tumor (due tothe presence of other biomarkers, metastases, etc.), but the tumorcannot be identified by traditional analytical techniques. All of thesemethods are features of the present invention.

The in vivo imaging and other diagnostic methods provided by the presentinvention are particularly useful in the detection of micrometastases ina human patient (e.g., a patient not previously diagnosed with cancer ora patient in a period of recovery/remission from a cancer).

In one embodiment, the present invention provides an in vivo imagingmethod wherein a target-specific protein of the present invention isconjugated to a detection-promoting radio-opaque agent, the conjugatedprotein is administered to a host, such as by injection into thebloodstream, and the presence and location of the labeled protein in thehost is assayed. Through this technique and any other diagnostic methodprovided herein, the present invention provides a method for screeningfor the presence of disease-related cells in a human patient or abiological sample taken from a human patient and/or for assessing thedistribution of target-specific protein prior to target-specific ADCtherapy.

For diagnostic imaging, radioisotopes may be bound to a target-specificprotein either directly or indirectly by using an intermediaryfunctional group. Useful intermediary functional groups includechelators, such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid (see for instance U.S. Pat. No.5,057,313).

In addition to radioisotopes and radio-opaque agents, diagnostic methodsmay be performed using target-specific proteins that are conjugated todyes (such as with the biotin-streptavidin complex), contrast agents,fluorescent compounds or molecules and enhancing agents (e.g.paramagnetic ions) for magnetic resonance imaging (MRI) (see, e.g., U.S.Pat. No. 6,331,175, which describes MRI techniques and the preparationof proteins conjugated to a MRI enhancing agent). Suchdiagnostic/detection agents may be selected from agents for use in MRI,and fluorescent compounds. In order to load a target-specific proteinwith radioactive metals or paramagnetic ions, it may be necessary toreact it with a reagent having a long tail to which a multiplicity ofchelating groups are attached for binding the ions. Such a tail may be apolymer such as a polylysine, polysaccharide, or another derivatized orderivatizable chain having pendant groups to which may be boundchelating groups such as, e.g., porphyrins, polyamines, crown ethers,bisthiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose. Chelates may be coupled to target-specific proteinsusing standard chemistries.

Thus, the present invention provides a diagnostic target-specificprotein, wherein the target-specific protein is conjugated to a contrastagent (such as for magnetic resonance imaging, computed tomography, orultrasound contrast-enhancing agent) or a radionuclide that may be, forexample, a gamma-, beta-, alpha-, Auger electron-, or positron-emittingisotope.

In a further aspect, the invention relates to a kit for detecting thepresence of target antigen or a cell expressing the target, in a sample,comprising:

-   -   a target-specific antibody of the invention; and    -   instructions for use of the kit.

In one embodiment, the present invention provides a kit for diagnosis ofcancer comprising a container comprising a target-specific protein, andone or more reagents for detecting binding of the target-specificprotein to the target. Reagents may include, for example, fluorescenttags, enzymatic tags, or other detectable tags. The reagents may alsoinclude secondary or tertiary antibodies or reagents for enzymaticreactions, wherein the enzymatic reactions produce a product that may bevisualized. In one embodiment, the present invention provides adiagnostic kit comprising one or more target-specific proteins of thepresent invention in labeled or unlabeled form in suitable container(s),reagents for the incubations for an indirect assay, and substrates orderivatizing agents for detection in such an assay, depending on thenature of the label. Control reagent(s) and instructions for use alsomay be included.

Diagnostic kits may also be supplied for use with a target-specificprotein, such as a labeled target-specific protein, for the detection ofthe presence of the target in a tissue sample or host. In suchdiagnostic kits, as well as in kits for therapeutic uses describedelsewhere herein, a target-specific protein typically may be provided ina lyophilized form in a container, either alone or in conjunction withadditional antibodies specific for a target cell or peptide. Typically,a pharmaceutically acceptable carrier (e.g., an inert diluent) and/orcomponents thereof, such as a Tris, phosphate, or carbonate buffer,stabilizers, preservatives, biocides, inert proteins, e.g., serumalbumin, or the like, also are included (typically in a separatecontainer for mixing) and additional reagents (also typically inseparate container(s)). In certain kits, a secondary antibody capable ofbinding to the target-specific protein, which typically is present in aseparate container, is also included. The second antibody is typicallyconjugated to a label and formulated in a manner similar to thetarget-specific protein of the present invention. Using the methodsdescribed above and elsewhere herein, target-specific proteins may beused to define subsets of cancer/tumor cells and characterize such cellsand related tumor tissues.

Sequences

SEQ ID NO: Clone name Sequence SEQ ID NO: 1 huCD3 VH CDR1 GFTFNTYASEQ ID NO: 2 huCD3 VH CDR2 IRSKYNNYAT SEQ ID NO: 3 huCD3 VH CDR3VRHGNFGNSYVSWFAY SEQ ID NO: 4 huCD3 VL CDR1 TGAVTTSNY huCD3 VL CDR2 GTNSEQ ID NO: 5 huCD3 VL CDR3 ALWYSNLWV SEQ ID NO: 6 huCD3 VH1EVKLVESGGGLVQPGGSLRLSCAASGFTFNTY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMY YCVRHGNFGNSYVSWFAYWGQGTLVTVSSSEQ ID NO: 7 huCD3 VH2 EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 8 huCD3 VH3EVKLVESGGGLVKPGRSLRLSCAASGFTFNTY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMY YCVRHGNFGNSYVSWFAYWGQGTLVTVSSSEQ ID NO: 9 huCD3 VH4 EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTMVTVSS SEQ ID NO: 10 huCD3 VL1QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTS NYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSN LWVFGGGTKLTVL SEQ ID NO: 11 huCD3 VL2QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTS NYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQADDESIYFCALWYSN LWVFGGGTKLTVL SEQ ID NO: 12 huCD3 VL3QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTS NYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQADDESDYYCALWYSN LWVFGGGTKLTVL SEQ ID NO: 13IgG1m(f) heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY chainFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS constantLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region RVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 14 Mature human QDGNEEMGGITQTPYKVSISGTTVILTCPQYPCD3ε (epsilon) GSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRAR VCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPP PVPNPDYEPIRKGQRDLYSGLNQRRISEQ ID NO: 15 Human CD3δ MEHSTFLSGLVLATLLSQVSPFKIPIEELEDR (delta)VFVNCNTSITWVEGTVGTLLSDITRLDLGKRI LDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGH ETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK SEQ ID NO: 16 VH huCLB-T3/4 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMFWVRQAPGKGLEWVATISRYSRYIYYPDSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRPLYGSSPDYWGQGTLVTVSS SEQ ID NO: 17 VL huCLB-T3/4EIVLTQSPATLSLSPGERATLSCSASSSVTYV HWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCFQGSGYPLT FGSGTKLEMR SEQ ID NO: 18 VH HER2 169QVQLVQSGAEVKKPGASVKVSCKASGYTFTNY GISWVRQAPGQGLEWMGWLSAYSGNTIYAQKLQGRVTMTTDTSTTTAYMELRSLRSDDTAVYYC ARDRIVVRPDYFDYWGQGTLVTVSS SEQ ID NO: 19VL HER2 169 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSG SGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIK SEQ ID NO: 20 IgG1m(f)- ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYLFLEDA heavy FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS chain constantLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK region RVEPKSCDKTHTCPPCPAPE FEGGPSVFLFPP KPKDTLMISRTPEVTCVVV A VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 21 Mature cynoQDGNEEMGSITQTPYQVSISGTTVILTCSQHL CD3ε (epsilon)GSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSG YYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAK AKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI

EXAMPLES Example 1—Generation of Non-Activating AntibodiesNon-Activating Mutations

Several antibody variants were generated with one or more amino acidsubstitutions in the Fc region. A non-activating Fc region prevents theantibody from interacting with Fc-receptors present on blood cells, suchas monocytes, or with C1q to activate the classical complement pathway.Reduction of the Fc activity was tested in antibody variants thatcontain different combinations of amino acid substitutions in the Fcregion. Maximally five amino acid substitutions were introduced, whichinclude the mutations N297Q, L234A, L235A, L234F, L235E, D265A, andP331S. Substitutions in one or more of these five amino acid positionswere introduced in the K409R and F405L IgG1 backbone. The followingFc-domain variants were generated: NQ (refers to the N297Qsubstitution), LFLE (refers to the L234F/L235E substitutions), LALA(refers to the L234A/L235A substitutions), LFLENQ (refers to theL234F/L235E/N297Q substitutions), LFLEDA (refers to theL234F/L235E/D265A substitutions), DA (refers to the D265A substitution),DAPS (refers to the D265A/P331S substitutions), DANQ (refers to theD265A/N297Q substitutions), LFLEPS (refers to the L234F/L235E/P331Ssubstitutions), and LFLEDANQPS (refers to theL234F/L235E/D265A/N297Q/P331S substitutions).

CD3 Antibodies

Various CD3 antibodies were used in monospecific and bispecific format.

In some examples the heavy and light chain variable region sequences ofhuCLB-T3/4 (SEQ ID NOs: 16 and 17, respectively) were used, which is ahumanized version of the murine antibody CLB-T3/4. Both sequences werecloned into the relevant expression vectors and expressed byco-transfection in HEK293F cells.

In some examples humanized variant (VH according to SEQ ID NO:8 and VLaccording to SEQ ID NO:10) of a murine CD3 antibody (described as“huCD3”) as described in U.S. Pat. No. 8,236,308 were used. Humanizationof this CD3 antibody was performed by Antitope (Cambridge, UK) usingtheir improved version of the germline humanization (CDR-grafting)technology, as described in EP 0 629 240. Using this technology, 4different VH chains (SEQ ID NOs.:6, 7, 8, and 9) and 3 different VLchains (SEQ ID NOs.:10, 11, and 12) were designed.

HER2 Antibody

In some of the examples an antibody against HER2 was used. The VH and VLsequences for this HER2-specific antibody (VH HER2 169 and VL Her2 160SEQ ID NOs.:18 and 19, respectively) as described in WO2012143524[Genmab]; and Labrijn et al., PNAS 2013, 110: 5145-50.

b12 Antibody

In some of the examples the antibody b12, a gp120 specific antibody(Barbas, C F. J Mol Biol. 1993 Apr. 5; 230(3):812-23.) was used as anegative control.

Expression

Antibodies were expressed as IgG1,κ or IgG1,λ with or without thenon-activating mutations described above and being additionally modifiedin their Fc regions as follows: IgG1-HER2-K409R, IgG1-b12-K409R,IgG1-CD3-F405L. Plasmid DNA mixtures encoding both heavy and light chainof antibodies were transiently transfected to Freestyle HEK293F cells(Invitrogen, US) using 293fectin (Invitrogen, US) essentially asdescribed by the manufacturer.

Purification of Antibodies

Culture supernatant was filtered over 0.2 μm dead-end filters, loaded on5 mL MabSelect SuRe columns (GE Health Care) and eluted with 0.1 Msodium citrate-NaOH, pH 3. The eluate was immediately neutralized with2M Tris-HCl, pH 9 and dialyzed overnight to 12.6 mM NaH2PO4, 140 mMNaCl, pH 7.4 (B. Braun). Alternatively, subsequent to purification, theeluate was loaded on a HiPrep Desalting column and the antibody wasexchanged into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun) buffer.After dialysis or exchange of buffer, samples were sterile filtered over0.2 μm dead-end filters. Purity was determined by SDS-PAGE andconcentration was measured by absorbance at 280 nm. Purified antibodieswere stored at 4° C.

Generation of Bispecific Antibodies

Bispecific antibodies were generated in vitro according to the DuoBody®technology platform, i.e. 2-MEA-induced Fab-arm exchange as described inWO 2011/147986 and Labrijn et al. (Labrijn et al., PNAS 2013, 110:5145-50; Gramer et al., MAbs 2013, 5: 962-973). The basis for thismethod is the use of complementary CH3 regions, which promote theformation of heterodimers under specific assay conditions. To enable theproduction of bispecific antibodies by this method, IgG1 moleculescarrying certain mutations in the CH3 region were generated: in one ofthe parental IgG1 antibody the F405L mutation, in the other parentalIgG1 antibody the K409R mutation. To generate bispecific antibodies,these two parental antibodies, each antibody at a final concentration of0.5 mg/mL, were incubated with 25 or 75 mM 2-mercaptoethylamine-HCl(2-MEA) in a total volume of 500 μL TE at 31° C. for 5 hours. Thereduction reaction was stopped when the reducing agent 2-MEA is removedby using PD-10 columns (GE-healthcare, product #17-0851-01),equilibrated with 25 mL PBS. Prior to desalting, 2 mL PBS (B. Braun,product #3623140) was added to the samples to adjust the volume to 2.5mL. Elution was done in 3.5 mL PBS. Samples were collected in AmiconUltra centrifugal units (30 kD MWCO, Millipore, product # UFC803096) andconcentrated by centrifuging 8 min at 3000×g. Volumes were adjusted to500 μL (when needed) with PBS and samples were sterile-filtered over a0.2 μm filter (Millex-GV, product # SLGV004SL). The bispecific productswere stored at 2-8° C.

Example 2—Binding of Antibody Mutants to Jurkat or AU565 Cells

Binding of purified variants of antibodies IgG1-CD3 (huCLB-T3/4,containing the F405L mutation), IgG1-HER2 (HER2-169, containing theK409R mutation), and bispecific (bs)IgG-CD3×HER2 molecules withadditional mutations in the Fc-domain (see Example 1) to CD3-positiveJurkat cells or HER2-positive AU565 cells was analyzed by FACS analysis.Cells (1×105 cells/well) were incubated in polystyrene 96-wellround-bottom plates (Greiner bio-one 650101) with serial dilutions ofantibody preparations (range 2 to 10000 ng/mL in 4-fold dilutions forJurkat cells and range 1 to 3000 ng/mL in 4-fold dilutions for on AU565cells) in 100 μL PBS/0.1% BSA/0.02% azide at 4° C. for 30 min.

After washing twice in PBS/0.1% BSA/0.02% azide, cells were incubated in100 μL with secondary antibody at 4° C. for 30 min. As a secondaryantibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab′)2(109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove,Pa.) diluted 1/100 in PBS/0.1% BSA/0.02% azide, was used for allexperiments. Next, cells were washed twice in PBS/0.1% BSA/0.02% azide,resuspended in 150 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACSCantoll (BD Biosciences). Binding curves were analyzed using non-linearregression (sigmoidal dose-response with variable slope) using GraphPadPrism V5.04 software (GraphPad Software, San Diego, Calif., USA).

Binding of IgG1-CD3 and bsIgG1-CD3×HER2 antibody variants to Jurkatcells was not affected by the introduction of the indicated mutations inthe Fc-domain and was identical for all tested mutants and wild typeantibody (FIGS. 1A and 1B and FIGS. 2A and 2B).

Similarly, binding of IgG1-HER2 and bsIgG1-CD3×HER2 antibody variants toAU565 cells was not affected by the introduction of the indicatedmutations in the Fc-domain and was identical for all tested mutants andwild type antibody (FIGS. 1C and 1D and FIGS. 2C and 2D).

Example 3—CD69 Expression on T-Cells in PBMC Culture

CD69 expression on T-cells was evaluated by FACS analysis to determineearly activation of T-cells after incubation with IgG1-CD3 antibodieswith mutations in the Fc-domain (see Example 1).

PBMCs were isolated from whole blood or buffy coat by density gradientseparation using Leucosep tubes (#227290; Greiner Bio-one, Alphen a/dRijn, The Netherlands), washed with PBS and resuspended in culturemedium.

A dose response series of IgG1-CD3 antibody variants, a negative control(IgG1-CD3 Fab) and positive control (IgE-CD3) were prepared in culturemedium (ranging from 1 to 1000 ng/mL in 3-fold dilutions) and added tothe wells of a 96-well round bottom plate containing the PBMCs. After16-24 hours incubation, cells were pelleted by centrifugation andsupernatant (containing cytokines) collected and stored at −20° C. Cellswere then washed with PBS/0.1% BSA/0.02% azide and stained for 30minutes at 4° C. with a mouse-anti-human CD28-PE (854.222.010; Sanquin,Amsterdam, The Netherlands; T-cell marker) and mouse-anti-human CD69-APCantibody (340560; BD Biosciences, Franklin Lakes, N.J.). Unboundantibodies were removed by washing twice with PBS/0.1% BSA/0.02% azide.Cells were resuspended in 150 μL/well and CD69-expression on CD28positive cells was measured on FACS Canto II (BD Biosciences).

FIG. 3 shows that CD69 expression was high on cells which were incubatedwith IgG1-CD3, IgG1-CD3-DA and IgG1-CD3-DAPS. Incubation withIgG1-CD3-N297Q and IgG1-CD3-LALA induced somewhat lower expressionlevels of CD69 compared to wild type IgG1-CD3, and incubation withIgG1-CD3-LFLE and IgG1-CD3-LFLEPS induced CD69 to a lesser extent.Incubation of PBMCs with IgG1-CD3 Fab, IgG1-b12, IgG1-CD3-LFLEDA,IgG1-CD3-LFLENQ, IgG1-CD3-DANQ and IgG1-CD3-LFLEDANQPS antibodies didnot induce any expression of CD69 on T-cells.

Example 4—CD3 Antibody-Induced T-Cell Proliferation

The effect of CD3 antibody variants (described in Example 1) on theproliferation of T-cells was evaluated by the Cell proliferation ELISAkit from Roche Applied Science (Cell Proliferation ELISA, BrdU kit,#11647229001; Roche Applied Science, Mannheim, Germany), which wasperformed according to the manufacturer's instructions.

PBMCs, isolated from whole blood or buffy coat, were incubated in96-well culture plates with dilution series (ranging from 0.1 to 1000ng/mL) of IgG1-CD3 variants. IgE-CD3 and IgG1-CD3 were included aspositive controls and IgG1-b12 (with K409R mutation for generation ofbispecific antibodies) as a negative control. After 3 days of incubationwith the antibodies, BrdU (Roche Applied Science, Mannheim, Germany) wasadded to the medium and plates were incubated for 5 hours. Cells werethen pelleted by centrifugation and supernatant collected and stored at−20° C. Plates were dried and stored at 4° C. until ELISA was performed.

BrdU incorporation in the DNA was determined by ELISA according to themanufacturer's instructions (Cell Proliferation ELISA, BrdU kit,#11647229001; Roche Applied Science). Cells were fixed to the plates,where after the plates were incubated for 90 minutes at room temperature(RT) with an anti-BrdU antibody conjugated with peroxidase. Plates werewashed with PBST and binding was detected using ABTS buffer (instead ofthe TMB solution provided with the kit). Color development was stoppedafter 30 min by adding 2% oxalic acid to the wells. OD405 nm was thenmeasured on an EL808 ELISA-reader.

FIG. 4 shows that incubation of PBMCs with IgG1-CD3, IgG1-CD3-DA andIgG1-CD3-DAPS induced strong proliferation of T-cells, even at very lowconcentrations of antibody. Incubation with IgG1-CD3-N297Q orIgG1-CD3-LALA induced dose-dependent proliferation, which was comparableto the IgE-CD3 positive control. Incubation of PBMCs with IgG1-CD3 Fab,IgG1-b12, IgG1-CD3-LFLE, IgG1-CD3-LFLEDA, IgG1-CD3-LFLENQ,IgG1-CD3-LFLEPS, IgG1-CD3-DANQ and IgG1-CD3-LFLEDANQPS antibodies didnot induce proliferation of T-cells.

Based on the results from Example 3 and 4, a subset of mutants that wereconsidered least activating, was subjected to further analysis.

Example 5—In Vitro T-Cell-Mediated Cytotoxicity Induced byNon-Activating Antibody Variants

AU565 (human breast carcinoma) cells were cultured in RPMI 1640supplemented with 10% (vol/vol) heat inactivated CCS, 1.5 g/L sodiumbicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g/L glucose (Sigma), 50IU/mL penicillin, and 50 μg/mL streptomycin. The cell line wasmaintained at 37° C. in a 5% (vol/vol) CO₂ humidified incubator. AU565cells were cultured to near confluency. Cells were trypsinized,re-suspended in culture medium and passed through a cell strainer toobtain a single cell suspension. 5×10⁴ cells were seeded in each well ofa 96-well culture plate, and cells were incubated at least 3 hrs. at 37°C., 5% CO2 to allow adherence to the plate.

Peripheral blood mononuclear cells (PBMC) were isolated from blood fromhealthy volunteers using Leucosep 30 mL tubes, according to themanufacturer's protocol (Greiner Bio-one). Isolated PBMCs were washedwith PBS, re-suspended in culture medium and added in a 1:1 ratio to theAU565 tumor cells in the 96-well plates. The percentage of T-cellspresent in PBMCs was measured by FACS-analysis, using a mouse anti-humanCD3-PerCP (BD, #345766) antibody (for staining T-cells). The T-cellcontent in the population of used PBMCs was typically 50 to 60%.

Dilution series (final concentrations ranging from 0.004 to 1000 ng/mL)of IgG1-b12, IgG1-CD3, IgG1-HER2, and bispecific CD3×b12 and CD3×HER2antibodies expressed as different Fc-variants, wild type, N297Q, LFLE,LALA, LFLENQ, LFLEDA, DANQ, and LFLEDENQPS, were prepared in culturemedium and added to the plates. Plates were incubated for 3 days at 37°C., 5% CO₂. Incubation of cells with 1 μM staurosporin (#56942-200,Sigma) was used as reference for 100% tumor cell kill. After incubation,supernatants were removed and stored at −20° C. for later analysis ofcytokine release (see Example 6). Plates were washed twice with PBS, and150 μL culture medium containing 10% Alamar blue was added to each well.Plates were incubated for 4 hours at 37° C., 5% CO₂. Absorbance at 590nm was measured (Envision, Perkin Elmer, Waltham, Mass.).

Two experiments were performed using PBMCs from different donors. In thefirst experiment Fc-variants N297Q, LFLE, LFLENQ, LFLEDA, DANQ, andLFLEDANQPS were tested (FIG. 5A-G). In the second experiment Fc-variantsLFLEDA and LALA were tested (FIG. 6A-C). Antibodies with wild-typeFc-domains were included in both experiments as reference. Incubationwith wild-type monospecific IgG1-CD3 or bispecific CD3×b12 antibodiesinduced unspecific killing of target cells (FIGS. 5A-G and 6A-C). Themonospecific IgG1-CD3 and bsIgG1-CD3×b12 variants N297Q (FIG. 5A-G) andLALA (FIG. 6A-C) still induced some unspecific target cell killing,albeit to a lesser extent than the wild-type antibody tested in the sameexperiment. Unspecific killing was not induced by any of the othertested IgG1-CD3 or bsIgG1-CD3×b12 antibodies with non-activatingmutations (FIGS. 5A-G and 6A-C).

All bispecific CD3×HER2 antibodies induced dose-dependent killing ofAU565 cells with at least comparable efficacy compared to the wild typebispecific CD3×HER2 antibody without non-activating mutations (FIGS.4A-G and 6A-C). Maximum killing occured at very low concentrations.

No cytotoxicity was induced by wild-type or non-activating variants ofthe monospecific b12 or HER2 antibodies (FIGS. 4A-G and 6A-C).

Example 6—Cytokine Release Induced by Non-Activating Antibody Variants

Cytokines present in supernatant samples from cytotoxicity assays asperformed in Example 5 were quantified using the Pro-inflammatory kit(MSD, # K15007B-1).

In short, supernatant and calibrator samples were added to the multiplexplates and incubated for 1-2 hours at room temperature. Subsequently, lxDetection Antibody Solution, which was provided with the kit, was addedto the wells and incubated for another 1-2 hrs. The plates were washed 3times with PBST, Read Buffer T was added to the wells andchemiluminescence was measured on an imager. Cytokine concentrationswere calculated using the standard curves obtained from the calibratorsamples.

The results of the production of 9 cytokines (IFNγ, TNFα, GM-CSF, IL-1β,IL-2, IL-6, IL-8, IL-10, IL-12) are shown in FIG. 7A-I. Wild-typeIgG1-CD3 induced production of all 9 tested cytokines in variousamounts. Incubation of target and effector cells with monospecificIgG1-CD3 antibody variants LFLENQ, LFLEDA, DANQ, and LFLEDANQPS did notlead to substantial production of IFNγ, TNFα, GM-CSF, IL-1β, IL-2, IL-6,and IL-10, and production of small amounts of IL-8 and IL-12. Incubationof target and effector cells with monospecific IgG1-CD3-LALA variantinduced production high amounts of IL-8, production of low amounts of 7cytokines (IFNγ, TNFα, GM-CSF, IL-1β, IL-6, IL-10, IL-12), and nosubstantial amounts of IL-2.

Bispecific IgG1-CD3×HER2 antibodies, both wild-type and non-activatingvariants, induced production of all 9 cytokines (FIG. 7A-I). Cytokineproduction induced by bispecific IgG1-CD3×HER2 antibodies was somewhathigher compared to the cytokine production induced by the wild-typemonospecific IgG1-CD3 control, with the exception of production of IL-1βand IL-2.

Example 7—Evaluation of Binding of C1q to Non-Activating AntibodyVariants

Interaction of C1q with antibodies bound to a target cell is the firststep in the classical pathway of complement activation. Since wild-typeIgG1 harbors the interaction site for C1q, the interaction of C1q tothese non-activating IgG1 variants by an ELISA was evaluated.

Dilution series (range 7-30000 ng/mL in 4-fold dilutions) of IgG1-CD3,bsIgG1-CD3×HER2 and IgG1-CD20 (positive control) and non-activatingantibody variants as described above in Example 1 thereof were coated on96-well Microlon ELISA plates (Greiner, Germany) overnight at 4° C.Plates were washed and blocked with PBS supplemented with 0.025% Tween20 and 0.1% gelatine. With washings in between incubations, plates weresequentially incubated with 3% pooled human serum (Sanquin, product #M0008) for 1 h at 37° C., with 100 μL/well rabbit anti-human C1q (DAKO,product # A0136, 1/4.000) for 1 h at RT, and with 100 μL/well swineanti-rabbit IgG-HRP (DAKO, P0399, 1:10.000) as detecting antibody for 1h at RT. Detection was performed by addition of 1 mg/mL 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim,Germany) for about 30 min. The reaction was stopped by the addition of100 μL 2% oxalic acid. Absorbance was measured at 405 nm in a microplatereader (Biotek, Winooski, Vt.). Log transformed data were analyzed byfitting sigmoidal dose-response curves with variable slope usingGraphPad Prism software.

C1q showed binding to the antibodies with wild-type IgG1 Fc regions,IgG1-CD20, IgG1-CD3 and bsIgG1 CD3×HER2 (FIG. 8). No binding of C1q wasdetected on all evaluated antibody variants with non-activatingmutations (N297Q, LFLE, LFLENQ, LFLEDA, DA, DAPS, DANQ, LFLEPS,LFLEDANQPS, LALA) (FIG. 8).

Example 8—Binding of Non-Activating Antibody Variants to FcγRI

Binding of IgG1-CD3 antibody variants N297Q, LFLE, LFLEDA, LFLENQ, DANQ,LFLEDANQPS and LALA to the high affinity FcγRI expressed by IIa1.6 FcγRIcells was evaluated by FACS analysis.

IIa1.6 FcγRI cells (Van Vugt et al. Blood 1999, 94: 808-817) werecultured in RPMI medium supplemented with 10% Cosmic calf serum, 25mg/ml (55 mM) Methotrexate, 50 IU/mL penicillin, and 50 μg/mLstreptomycin. The cell line was maintained at 37° C. in a 5% (vol/vol)CO₂ humidified incubator.

IIa1.6 FcγRI cells (1×10⁵ cells/well) were incubated with serialdilutions of antibody preparations (range 10 to 10000 ng/mL in 4-folddilutions) for 30 min at 4° C. in polystyrene 96-well round-bottomplates (Greiner bio-one, #650101). Cells were washed with PBS/0.1%BSA/0.02% azide and stained for 30 minutes at 4° C. with R-Phycoerythrin(PE)-conjugated goat-anti-human IgG F(ab′)2 (109-116-098, JacksonImmunoResearch Laboratories) diluted 1/100 in PBS/0.1% BSA/0.02% azide.Then, cells were washed with PBS/0.1% BSA/0.02% azide, resuspended in150 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACS Cantoll (BDBiosciences). Binding curves were analyzed using non-linear regression(sigmoidal dose-response with variable slope) using GraphPad Prism V5.04software (GraphPad Software, San Diego, Calif., USA).

Wild-type IgG1-CD3 antibody showed strong binding to FcγRI (FIG. 9).IgG1-CD3 antibody variants N297Q and LALA showed weak binding to FcγRIon IIa1.6 FcγRI cells when tested at higher concentrations (>1000 ng/mL;FIG. 9). No substantial binding to IIa1.6 FcγRI cells was observed forantibody variants LFLE, LFLEDA, LFLENQ, DANQ and LFLEDANQPS (FIG. 9).

Example 9—Pharmacokinetic (PK) Analysis of Non-Activating AntibodyVariants

The mice in this study were housed in a barrier unit of the CentralLaboratory Animal Facility (Utrecht, The Netherlands) and kept infilter-top cages with water and food provided ad libitum. Allexperiments were approved by the Utrecht University animal ethicscommittee. 7-10 Weeks old C.B-17 SCID mice(C.B-17/Icr-Prkdc<Scid>/IcrIcoCrl, Charles-River) were injectedintravenously with 100 μg wild-type antibody (IgG1-CD3, IgG1-HER2, orbsIgG CD3×HER2) or non-activating variants thereof (LALA, LFLEDA,LFLENQ, DANQ or LFLEDANQPS) using 3 mice per group. 50 μL blood sampleswere collected from the saphenous vein at 10 minutes, 4 hours, 1 day, 2days, 7 days, 14 days and 21 days after antibody administration. Bloodwas collected into heparin containing vials and centrifuged for 5minutes at 10,000×g. Plasma was stored at −20° C. until determination ofantibody concentrations.

Human IgG concentrations were determined using a total hIgG sandwichELISA. For this assay, mouse mAb anti-human IgG-kappa clone MH16 (#M1268, CLB Sanquin, The Netherlands), coated to 96-well Microlon ELISAplates (Greiner, Germany) at a concentration of 2 μg/mL was used ascapturing antibody. After blocking plates with PBS supplemented with0.2% bovine serum albumin, samples were added, serially diluted withELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serumalbumin), and incubated on a plate shaker for 1 h at room temperature(RT). Plates were subsequently incubated with goat anti-human IgGimmunoglobulin (#109-035-098, Jackson, West Grace, Pa.) and developedwith 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS;Roche, Mannheim, Germany). The reaction was stopped after 30 min byadding 2% oxalic acid to the wells. Absorbance was measured in amicroplate reader (Biotek, Winooski, Vt.) at 405 nm.

Plasma clearance rates (mL/day/kg) were calculated based on the areaunder the curve (AUC), according to the following equation:

${Plasma}\mspace{14mu} {clearance}{= \frac{{Dose}\mspace{11mu} ( {{µg}\text{/}{kg}} )}{{AUC}( {{µg}\text{/}{mL}\text{/}{day}} )}}$

Data analysis was performed using Graphpad prism software.

FIG. 10A shows that the plasma human IgG concentrations were lower forantibody variants N297Q, DANQ, LFLENQ, and LFLEDANQPS when compared towild-type antibodies. The human IgG concentrations in plasma forantibody variants LFLEDA and LALA were similar to those of wild-typeantibodies.

FIG. 10B shows that the plasma clearance rates of antibody variantsN297Q, DANQ and LFLENQ were 2 to 3-fold higher than that of wild-typeantibody. The clearance rate of antibody variant LFLEDANQPS was 3-5times higher than that of wild-type antibody. Plasma clearance rates ofantibody variants LFLEDA and LALA were similar to that of wild-typeantibody.

Example 10—Binding of Humanized CD3 Antibodies and Non-ActivatingVariants Thereof to Human and Cynomolgous T-Cell Lines

Binding of purified variants of humanized CD3 (huCD3) antibodies andbispecific (bs)IgG1-huCD3×HER2 molecules with or without LFLEDAmutations in the Fc-domain (see Example 1) to the human T-cell lineJurkat or the cynomolgous T-cell line HSC-F was analyzed by FACSanalysis. In addition to the non-activating mutations, LFLEDA antibodyvariants comprise F405L or K409R mutations as described in Example 1.

Cells (1×10⁵ cells/well) were incubated in polystyrene 96-wellround-bottom plates (Greiner bio-one 650101) with serial dilutions ofantibody preparations (range 5 to 10,000 ng/mL in 3-fold dilutions) in100 μL PBS/0.1% BSA/0.02% azide at 4° C. for 30 min.

After washing twice in PBS/0.1% BSA/0.02% azide, cells were incubated in100 μL with secondary antibody at 4° C. for 30 min. As a secondaryantibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgG F(ab′)2(109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove,Pa.) diluted 1/100 in PBS/0.1% BSA/0.02% azide, was used for allexperiments. Next, cells were washed twice in PBS/0.1% BSA/0.02% azide,resuspended in 150 μL PBS/0.1% BSA/0.02% azide and analyzed on a FACSCantoll (BD Biosciences). Binding curves were analyzed using non-linearregression (sigmoidal dose-response with variable slope) using GraphPadPrism V5.04 software (GraphPad Software, San Diego, Calif., USA).

FIG. 11A shows that binding to Jurkat cells of the IgG1-huCD3 variantsH1L1 (SEQ ID NOs:6 and 10, respectively), H1L2 (SEQ ID NOs:6 and 11,respectively), H1L3 (SEQ ID NOs:6 and 12), H3L3 (SEQ ID NOs: 8 and 12,respectively), and H4L1 (SEQ ID NOs:9 and 10, respectively) with wildtype Fc region and parental IgG1-CD3 and IgG1-huCD3-H3L1 with LFLEDAmutations was similar. Binding of IgG1-huCLB-3/4, included as positivecontrol, was strong to Jurkat cells in comparison with the IgG1-huCD3variants. No binding was observed for the negative control antibodyIgG1-b12. H1 refers to the variable heavy chain region VH1, V1 refers tothe variable light chain region VL1, and so forth.

FIG. 11B shows that bispecific antibody variants bsIgG1 CD3×HER2, bsIgG1CD3×b12-LFLEDA, and bsIgG1 huCD3-H3L1×HER2-LFLEDA also bind to Jurkatcells. The maximal binding values for these bispecific antibodies ishigher than the maximal binding values of the monospecific antibodies.The EC50 concentrations of the bispecific antibodies were 6 to 10-foldhigher. Again, no binding was observed for the negative control antibodyIgG1-b12.

FIG. 12A shows that binding of the IgG1-huCD3 variants H1L1, H1L2, H1L3,H3L3, and H4L1 (as described above) with wild-type Fc region andparental IgG1-CD3 and IgG1-huCD3-H3L1 with LFLEDA mutations to thecynomolgous T-cell line HSC-F was similar. No binding was observed forhuCLB-3/4, which does not cross-react with cynomolgous CD3, and thenegative control antibody IgG1-b12.

FIG. 12B shows that bispecific antibody variants bsIgG1 CD3×HER2 andbsIgG1 huCD3-H3L1×HER2-LFLEDA also bind to HSC-F cells. The maximalbinding values for these bispecific antibodies is higher than themaximal binding values of the monospecific anti-CD3 variants. The EC50concentrations of the bispecific antibodies were 10 to 12-fold higherthan that of the monospecific anti-CD3 antibodies. Again, no binding wasobserved for the negative control antibody IgG1-b12.

Example 11—T-Cell Activation by Humanized CD3 Antibody Variants

CD69 expression on T-cells was evaluated by FACS analysis to determineearly activation of T-cells after incubation with humanized CD3 (huCD3)antibody variants with and without LFLEDA mutations in the Fc region. Inaddition to the non-activating mutations, LFLEDA antibody variantscomprise F405L or K409R mutations as described in Example 10.

PBMCs were isolated from whole blood or buffy coat by density gradientseparation using Leucosep tubes (#227290; Greiner Bio-one, Alphen a/dRijn, The Netherlands), washed with PBS and re-suspended in culturemedium.

A dose response series of huCD3 antibody variants, a negative control(IgG1-b12) and positive controls (IgE-huCD3 and parental IgG1-CD3) wereprepared in culture medium (ranging from 0.1 to 1,000 ng/mL in 10-folddilutions) and added to the wells of a 96-well round bottom platecontaining human or cynomolgous PBMCs. After 16-24 hours incubation,cells were pelleted by centrifugation and supernatant (containingcytokines) collected and stored at −20° C. Cells were then washed withPBS/0.1% BSA/0.02% azide and stained for 30 minutes at 4° C. with amouse-anti-human CD28-PE (854.222.010; Sanquin, Amsterdam, TheNetherlands; T-cell marker) and mouse-anti-human CD69-APC antibody(340560; BD Biosciences, Franklin Lakes, N.J.). Unbound antibodies wereremoved by washing twice with PBS/0.1% BSA/0.02% azide. Cells werere-suspended in 150 μL/well and CD69-expression on CD28 positive cellswas measured on FACS Canto II (BD Biosciences).

FIG. 13 shows that parental IgG1-CD3 and humanized IgG1-huCD3 variantswith wild type Fc region induced similar levels of CD69 expression onT-cells from human (FIG. 13A) and cynomolgous (FIG. 13B) origin.Non-activating (LFLEDA) parental IgG1-CD3 and IgG1-huCD3-H3L1 variantsinduced low levels of CD69 expression in human T-cells. No expression ofCD69 was induced by the non-activating IgG1-huCD3 variants incynomolgous T-cells. The control antibody IgG1-b12 also did not induceexpression of CD69 in human or cynomolgous T-cells.

Example 12—T-Cell Proliferation Induced by Humanized CD3 AntibodyVariants

The effect of humanized CD3 (huCD3) antibody variants (described inExamples 1 and 10) on the proliferation of human and cynomolgous T-cellswas evaluated by the Cell proliferation ELISA kit from Roche AppliedScience (Cell Proliferation ELISA, BrdU kit, #11647229001; Roche AppliedScience, Mannheim, Germany), which was performed according to themanufacturer's instructions.

Human or cynomolgous PBMCs, isolated from whole blood or buffy coat,were incubated in 96-well culture plates with dilution series (rangingfrom 0.1 to 1,000 ng/mL in 10-fold dilutions) of huCD3 antibodyvariants. IgE-CD3 and IgG1-huCLB-T3/4 were included as positive controlsand IgG1-b12 as negative control. After 3 days of incubation with theantibodies, BrdU (Roche Applied Science, Mannheim, Germany) was added tothe medium and plates were incubated for 5 hours. Cells were thenpelleted by centrifugation and supernatant collected and stored at −20°C. Plates were dried and stored at 4° C. until ELISA was performed.

BrdU incorporation in the DNA was determined by ELISA according to themanufacturer's instructions (Roche Applied Science, see cat. numberspecified above). Cells were fixed to the plates, where after the plateswere incubated for 90 minutes at RT with an anti-BrdU antibodyconjugated with peroxidase. Plates were washed with PBST and binding wasdetected using ABTS buffer (instead of the TMB solution provided withthe kit). Color development was stopped after 30 min by adding 2% oxalicacid to the wells. OD405 nm was then measured on an EL808 ELISA-reader.

FIG. 14 shows that incubation of PBMCs with parental IgG1-CD3 andhumanized IgG1-huCD3 variants with wild-type Fc region induced strongproliferation of human (FIG. 14A) and cynomolgous (FIG. 14B) T-cells,even at very low concentrations of antibody. Incubation withnon-activating LFLEDA variants of the IgG1-huCD3 antibodies did notinduce proliferation of human or cynomolgous T-cells. Thus, although thenon-activating variants of the IgG1-huCD3 antibodies induced low levelsof CD69 expression in human T-cells (as shown in Example 11), noproliferation of human T-cells was induced by these non-activatingIgG1-huCD3 variants.

Example 13—In Vitro T-Cell-Mediated Cytotoxicity Induced by HumanizedCD3 Antibody Variants

AU565 (human breast carcinoma) cells were cultured in RPMI 1640supplemented with 10% (vol/vol) heat inactivated CCS, 1.5 g/L sodiumbicarbonate (Lonza), 1 mM sodium pyruvate, 4.5 g/L glucose (Sigma), 50IU/mL penicillin, and 50 μg/mL streptomycin. The cell line wasmaintained at 37° C. in a 5% (vol/vol) CO₂ humidified incubator. AU565cells were cultured to near confluency, after which cells weretrypsinized, re-suspended in culture medium and passed through a cellstrainer to obtain a single cell suspension. 5×10⁴ cells were seeded ineach well of a 96-well culture plate, and cells were incubated at least3 hrs. at 37° C., 5% CO2 to allow adherence to the plate.

Human or cynomolgous PBMCs were isolated from whole blood or buffy coat.Isolated PBMCs were washed with PBS, re-suspended in culture medium andadded in a 1:1 ratio to the AU565 tumor cells in the 96-well plates. Thepercentage of T-cells present in PBMCs was measured by FACS-analysis,using a mouse anti-human CD3-PerCP (BD, #345766) antibody (for stainingT-cells). The T-cell content in the population of used PBMCs wastypically 50 to 60%.

Dilution series (final concentrations ranging from 0.001 to 1,000 ng/mL)of bispecific antibody variants bsIgG1 CD3×HER2, bsIgG1 CD3×b12-LFLEDA,and bsIgG1 huCD3-H3L1×HER2-LFLEDA were prepared in culture medium andadded to the plates. IgG1-HER2-LFLEDA and IgG1-b12 were included ascontrols. In addition to the non-activating mutations, LFLEDA antibodyvariants comprise F405L or K409R mutations for preparation in bispecificformat (see Example 10). Plates were incubated for 3 days at 37° C., 5%CO₂. Incubation of cells with 1 μM staurosporin (#56942-200, Sigma) wasused as reference for 100% tumor cell kill. Plates were washed twicewith PBS, and 150 μL culture medium containing 10% Alamar blue was addedto each well. Plates were incubated for 4 hours at 37° C., 5% CO₂.Absorbance at 590 nm was measured (Envision, Perkin Elmer, Waltham,Mass.). Bispecific CD3×HER2-LFLEDA antibody variants (parental andhumanized H3L1 variant) induced killing of AU565 cells at lowconcentrations using human or cynomolgous effector cells (FIG. 15). TheCD3 bispecific antibody huCLB-T3/4×HER2-LFLEDA, which shows no crossreactivity with cynomolgous CD3, only induced killing of AU565 cellswhen human PBMCs were used (FIG. 15A). Thus, no killing of target cellswas observed when cynomolgous effector cells were used in the assay(FIG. 15B). Incubation with monospecific IgG1-b12 or IgG1-HER2-LFLEDA orbispecific CD3×b12-LFLEDA antibodies did not induce unspecific killingof target cells (FIG. 15).

1-32. (canceled)
 33. A method of treating a disease comprisingadministering to a subject in need thereof an effective amount of aprotein comprising a first polypeptide and a second polypeptide, whereinsaid first polypeptide and second polypeptide each comprises at least ahinge region, a CH2 region, and a CH3 region of an immunoglobulin heavychain, wherein in at least one of said first polypeptide and secondpolypeptide, the amino acids in the positions corresponding to positionsL234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D,respectively.
 34. The method according to claim 33, wherein in at leastone of said first polypeptide and second polypeptide, the amino acid inthe positions corresponding to positions L234, L235, and D265 in a humanIgG1 heavy chain are not L, L, and D, respectively, and wherein theamino acids in the positions corresponding to positions N297 and P331 ina human IgG1 heavy chain are not Q and S, respectively.
 35. The methodaccording to claim 33, wherein said first polypeptide and secondpolypeptide are a first heavy chain and a second heavy chain of animmunoglobulin, respectively.
 36. The method according to claim 33,wherein said first polypeptide and second polypeptide further comprise afirst binding region and a second binding region, respectively.
 37. Themethod according to claim 33, wherein said protein further comprises afirst light chain and a second light chain of an immunoglobulin, whereinsaid first light chain is connected with said first heavy chain viadisulfide bridges and said second light chain is connected with saidsecond heavy chain via disulfide bridges, thereby forming a firstbinding region and a second binding region, respectively.
 38. The methodaccording to claim 36, wherein at least one of said first binding regionand second binding region binds to CD3.
 39. The method according toclaim 36, wherein both said first binding region and second bindingregion bind to CD3.
 40. The method according to claim 33, wherein in atleast one of said first polypeptide and second polypeptide, the aminoacids in the positions corresponding to positions L234, L235 and D265 ina human IgG1 heavy chain are hydrophobic or polar amino acids.
 41. Themethod according to claim 33, wherein in at least one of said firstpolypeptide and second polypeptide, the amino acids in the positionscorresponding to positions L234, L235 and D265 in a human IgG1 heavychain are aliphatic uncharged, aromatic, or acidic amino acids.
 42. Themethod according to claim 33, wherein in at least one of said firstpolypeptide and second polypeptide, the amino acids corresponding topositions L234, L235 and D265 in a human IgG1 heavy chain are F, E, andA; or A, A, and A, respectively.
 43. The method according to claim 33,wherein the isotype of the immunoglobulin heavy chain is selected fromthe group consisting of IgG1, IgG2, IgG3, and IgG4.
 44. The methodaccording to claim 39, wherein at least said first binding region isselected from the group consisting of: a. a binding region comprising aheavy chain variable region comprising the amino acid sequence set forthin SEQ ID NO: 6 and a light chain variable region comprising the aminoacid sequence set forth in SEQ ID NO: 12; b. a binding region comprisinga heavy chain variable region comprising the amino acid sequence setforth in SEQ ID NO: 8 and a light chain variable region comprising theamino acid sequence set forth in SEQ ID NO: 12; and c. a binding regioncomprising a heavy chain variable region comprising the amino acidsequence set forth in SEQ ID NO: 9 and a light chain variable regioncomprising the amino acid sequence set forth in SEQ ID NO:
 12. 45. Themethod according to claim 36, wherein said first binding region binds adifferent target than said second binding region.
 46. The methodaccording to claim 45, wherein the target to which the first bindingregion binds is present on different cells than the target to which thesecond binding region binds.
 47. The method according to claim 33,wherein in said first polypeptide, at least one of the amino acids inthe positions corresponding to a position selected from the groupconsisting of T366, L368, K370, D399, F405, Y407, and K409 in a humanIgG1 heavy chain has been substituted, and in said second polypeptide,at least one of the amino acids in the positions corresponding to aposition selected from the group consisting of: T366, L368, K370, D399,F405, Y407, and K409 in a human IgG1 heavy chain has been substituted,and wherein said substitutions of said first polypeptide and said secondpolypeptide are not in the same positions.
 48. The method according toclaim 47, wherein the amino acid in the position corresponding to F405in a human IgG1 heavy chain is L in said first polypeptide, and theamino acid in the position corresponding to K409 in a human IgG1 heavychain is R in said second polypeptide, or vice versa.
 49. The methodaccording to claim 33, wherein the protein is an antibody.
 50. Themethod according to claim 33, wherein the protein is a bispecificantibody.
 51. The method according to claim 36, wherein in both saidfirst polypeptide and second polypeptide, the amino acids in thepositions corresponding to L234, L235 and D265 in a human IgG1 heavychain are F, E, and A, respectively, said first binding region bindsCD3, and said second binding region binds a cancer-specific target. 52.The method according to claim 33, wherein the disease is selected fromthe group consisting of cancer, infectious disease, and autoimmunedisease.